PA1-913

antibody from Invitrogen Antibodies

Targeting: CASQ1 CASQ, PDIB1

Provider product page for PA1-913

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [141]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [2]
Immunohistochemistry [3]
Other assay [67]

Submit

Product number: PA1-913 - Provider product page
Provider: Invitrogen Antibodies
Product name: Calsequestrin Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Purifed from natural sources
Description: PA1-913 detects calsequestrin from canine, human, mouse, rat and sheep tissues. This antibody recognizes both cardiac and skeletal muscle calsequestrin.
Concentration: Conc. Not Determined

CASQ1 protein structure - PA1-913 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Calsequestrin was performed by loading 25 µg of RD (Lane 1), L6 (Lane 2) and mouse heart (Lane 3) cell lysates onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4ºC overnight. The membrane was probed with a Calsequestrin polyclonal antibody (Product # PA1-913) at a dilution of 1:5000 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Results show a band at approx. 55 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-Calsequestrin Polyclonal Antibody (Product # PA1-913) and a 45 kDa band corresponding to Calsequestrin-1 was observed across tissues tested . Tissue extracts (30 µg lysate) of L6 (Lane 1), RD (Lane 2), Mouse Skeletal Muscle (Lane 3), Rat Skeletal Muscle (Lane 4), Rat Heart (Lane 5), Mouse Liver (Lane 6), Rat Liver (Lane 7), Mouse Kidney (Lane 8) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0301BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Calsequestrin (green) showing staining in the cytoplasm and nucleus of C2C12 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Calsequestrin polyclonal antibody (Product # PA1-913) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Calsequestrin (green) showing staining in the cytoplasm and nucleus of C2C12 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Calsequestrin polyclonal antibody (Product # PA1-913) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Calsequestrin showing positive staining in the cytoplasm of paraffin-treated Human heart tissue (right) compared with a negative control in the absence of primary antibody (left). To expose target proteins, antigen retrieval method was performed using 10mM sodium citrate (pH 6.0) microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Calsequestrin polyclonal antibody (Product # PA1-913) diluted by 3% BSA-PBS at a dilution of 1:200 overnight at 4°C in a humidified chamber. Tissues were washed extensively PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Calsequestrin showing positive staining in the cytoplasm of paraffin-treated Human skeletal muscle (right) compared with a negative control in the absence of primary antibody (left). To expose target proteins, antigen retrieval method was performed using 10mM sodium citrate (pH 6.0) microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Calsequestrin polyclonal antibody (Product # PA1-913) diluted by 3% BSA-PBS at a dilution of 1:200 overnight at 4°C in a humidified chamber. Tissues were washed extensively PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Calsequestrin showing positive staining in the cytoplasm of paraffin-treated Mouse heart tissue (right) compared with a negative control in the absence of primary antibody (left). To expose target proteins, antigen retrieval method was performed using 10mM sodium citrate (pH 6.0) microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Calsequestrin polyclonal antibody (Product # PA1-913) diluted by 3% BSA-PBS at a dilution of 1:200 overnight at 4°C in a humidified chamber. Tissues were washed extensively PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Ca 2+ handling proteins. (A) Representative western blot images of Ca V 1.1, RyR1, SERCA1, SERCA2, calsequestrin (1 and 2), and sarcolipin. To allow the use of data in multiple western blots each band within a single western blot was normalized to GAPDH for that sample as a loading control and then normalized to the average WT values for that particular gel to give %WT. (B) Analysis of muscle levels of Ca V 1.1 normalized to GAPDH. (C) Analysis of muscle levels of RyR1 normalized to GAPDH. (D) Analysis of muscle levels of SERCA1 normalized to GAPDH. (E) Analysis of muscle levels of SERCA2 normalized to GAPDH. (F) Analysis of muscle levels of CSQ normalized to GAPDH. (G) Analysis of sarcolipin normalized to GAPDH. (H) SERCA activity as a function of Ca 2+ concentration. (I) Scheme of changes in Ca 2+ handling proteins. Values are shown as mean +- SEM. * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2--figure supplement 2. Analysis of RNA and protein expression levels of the major cardiac Ca 2+ -handling proteins in PLN/DWORF Tg mice. ( A ) RNA levels of the indicated genes as quantified by qRT-PCR in 16-week-old heart tissue. Atp2a2 , SERCA2a; Ryr2 , ryanodine receptor 2; Cacna1c, alpha1C-subunit of the L-type Ca 2+ channel; Casq2 , calsequestrin 2. Data are normalized to 18S and presented as expression level relative to WT, mean +-SD for n = 10 mice per genotype. ( B ) Representative immunoblots of cardiac homogenates from mice with the indicated genotypes. PS16, phospho-serine 16 on PLN; PT17, phospho-threonine 17 on PLN; tPLN, total phospholamban; RyR2, ryanodine receptor 2; LTCC, L-type Ca 2+ channel (alpha1C-subunit); Casq2, calsequestrin 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ( C ) Western blots for n = 9-10 mice of each genotype were quantified using ImageJ and data are normalized to GAPDH and expressed as mean +-SD relative to WT. ( D ) Quantification of total phospholamban and its phosphorylation status as assessed by western blot ( B ) and expressed as relative to WT. Western blots were quantified with ImageJ software. Phosphorylation blots (PS16 and PT17) were normalized to total PLN (tPLN). Total PLN was normalized to GAPDH. Data are expressed as mean +-SD for n = 9-10 mice per genotype. Statistical comparisons between groups were evaluated by Student's t-test. p-value *p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6--figure supplement 1. Expression and post-translational modifications of Ca 2+ -handling proteins. ( A ) Immunoblots of cardiac homogenates from mice with the indicated genotypes. ( B ) Immunoblots were quantified using ImageJ and normalized to GAPDH. LTCC, L-type Ca 2+ channel (alpha1C-subunit); Casq2, calsequestrin 2; RyR2, ryanodine receptor 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ( C ) Quantification of the phosphorylation status and oligomerization of phospholamban as assessed by western blot (panel A ) and expressed as relative to WT. Western blots were quantified with ImageJ software. Phosphorylation blots (PS16 and PT17) were normalized to total PLN (tPLN). Total PLN was normalized to GAPDH. tPLN, total phospholamban; PS16, phospho-serine 16 on PLN; PT17, phospho-threonine 17 on PLN. Data are expressed as mean +-SD for n = 4-6 mice per genotype. ( D ) RNA levels of the indicated genes as quantified by qRT-PCR in 8-week-old heart tissue. Atp2a2 , SERCA2a; Cacna1c, alpha1C-subunit of the L-type Ca 2+ channel; Casq2 , calsequestrin 2; Pln , phosholamban; Ryr2 , ryanodine receptor 2. Data are normalized to 18S values and presented as expression level relative to WT, mean +-SD for n = 4-5 mice per genotype.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: FIGURE 8 Molecular determinants of ryanodine receptor type-2 (RyR2) dysfunction. (A) Representative Western blot examples of total RyR2, Ser2808-, and Ser2814-phosphorylated RyR2 and calsequestrin (CSQ; left), as well as junctin and junctophilin-2 (JPH2; right) protein expression levels. GAPDH was used as loading control. (B) Quantification of total RyR2, Ser2808-, and Ser2814-phosphorylated RyR2, Ser2808/total RyR2, and Ser2814/total RyR2 protein expression levels in Ctl (white bars), HFrEF (blue bars) and HFrEF-cAF (red/blue-striped bars) patients. (C) Protein expression levels of the RyR2-interacting proteins CSQ, junctin, and JPH2 in Ctl, HFrEF and HFrEF-cAF patients. Data are shown relative to Ctl. Numbers in bars indicate number of patients. * indicates P < 0.05 vs. Ctl, # indicates P < 0.05 vs. HFrEF.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 Protein levels of sarcoplasmic reticulum Ca 2+ -handling proteins in intrinsic laryngeal muscles (ILM), cricothyroid (CT) muscle, extraocular muscles (EOM), and tibialis anterior (TA) muscle. Western blot analysis showing relative abundance of indicated proteins: calsequestrins 1 and 2 (CASQ1 and CASQ2), Sercas 1 and 2 (SERCA1 and SERCA2), calmodulin (CaM), calmodulin kinase II (CaMKII), and Orai1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control for protein loading, Western blot transfer and nonspecific changes in protein levels. The molecular weight, expressed in kDa, for each protein is indicated. Quantifications from three independent muscle samples, each sample containing muscles pooled from three or four different rats. Asterisks and different letter combination indicate statistical significance (* P < 0.05 and ab, ac, ad, bc, or bd P < 0.05, respectively). In ILM, CASQ 1 was more abundant than CASQ2 compared with TA. SERCA1 was less than SERCA2 in ILM and higher compared with TA. CT and the other ILM showed similar levels of the proteins studied.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 1 Effect of SERCA1b shRNA on the expression pattern of proteins involved in Ca 2+ -homeostasis. Examination of decreased SERCA1b expression in C2C12 myotubes. ( A-B ) Protein expression of SERCA was detected by Western-blot analysis to prove the efficiency of SERCA1b-specific shRNA in multinucleated myotubes. Different stably transfected clones, pool of the clones and scrambled shRNA transfected cells were compared. The 115 kDa isoforms were detected either by SERCA1b specific antibody corresponding to the terminal octamer of the protein or by an antibody recognizing both SERCA1a and b isoforms. Actin was used as a control. ( C ) Western blot analysis to detect the SR calcium-binding protein (calsequestrin) expression in selected cloneC1, C5, scrambled shRNA transfected, and parental cells. ( D ) Western-blot analysis showing the protein level of the key molecule of SOCE (STIM1). Total protein samples were used (30 mug in each lane) to examine the protein expression level. For preparing protein samples cultures were harvested on the 5 th day of differentiation in each cases. Representative data of 3 independent experiments.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Expression levels of selected proteins in gastrocnemius extracts from semistarved and refed rats and their respective controls were determined by Western blots . (A) Amount of myosin heavy chains (MyHC) were determined by Coomassie staining in 3 independents experiments. MyHC were similarly abundant in all 4 groups and were thus validated as internal controls for the quantification of the other muscle markers under study. Selected proteins that are well known markers of Ca 2+ handling in slow-twitch or fast-twitch fibers were analyzed: (B) SERCA1, (C) SERCA2, (D) calsequestrin 1, (E) calsequestrin 2, and (F) parvalbumin. The abundance of transcription factors that control the development of slow-twitch muscles either positively or negatively were also determined: (G) calcineurin, (H) PGC1-alpha (the most intense band at the expected size for the full-length protein was analyzed; the smaller molecular weight bands likely to be degradation products were excluded), and (I) FoxO1 (all bands, likely representing native, methylated, and acetylated forms, were quantified). The signals (B-I) were corrected for their MyHC content and normalized to the signal of a pool sample (a mixture of aliquots of all extracts) loaded on the gels for the purpose of intra-gel and inter-gel comparison. The signals from an extract of soleus muscle (a slow-twitch muscle), referred to as SOL, are shown for comparison. They were acquired under the same exposure condition as the experimental grou

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Comparison of protein expression in LV sample from control and cardiomyopathic patients. A: Top; representative SERCA2 and respective GAPDH stainings obtained in normal and cardiomyopathic LV samples. Bottom; Mean+-SEM, *** P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Correlative confocal and super-resolution imaging of a human cardiac tissue section. The section was ~10 um thick and was labeled with phalloidin for f-Actin (Alexa 488), WGA for the cell membrane and extracellular matrix (Alexa 594), along with antibodies for the ryanodine receptor (RyR, Alexa 680) and calsequestrin (CSQ, Alexa 750). In addition to the applied labelling, a strong endogenous fluorescence signal from lipofuscin was recorded. The shorter wavelength labels (Actin, WGA, and RyR) were imaged on a confocal microscope, and the sample then taken to the localisation microscope where super-resolution imaging of the longer wavelength labels (RyR, lipofuscin, CSQ) was performed. Panel A shows an overview of the cellular structure across a large tissue area that is indicated by the actin labeling (largely muscle cell contractile protein). Scale bar 100 um. Panel B is a projection of a confocal stack taken of the region indicated in A. Scale bar 10 um. Panel C shows a confocal stack of a small detail area from B and panel D shows an optically sectioned super-resolution stack, within the region covered by the confocal stack in C. Panels E & F compare corresponding confocal (F) and super-resolution (E) images both using the RyR-Alexa 647 signal. Note the good correlation between the data and the improvement in resolution in E. Scale bar 1 um. G : 3-colour super-resolution image of a small area in the tissue sample, note the improved resolution as compared to the con

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Expression level of skeletal muscle proteins, the amount of releasable Ca 2+ from the SR to the cytosol, and the resting cytosolic Ca 2+ level in primary skeletal myotubes. ( a ) The immunoblot analysis of proteins mediating Ca 2+ movements and handling in skeletal muscle was conducted using the lysate of myotubes treated with sildenafil. Fifteen proteins were examined, and no expression levels were changed by sildenafil (the expression level of proteins is presented as bar graphs in Supplementary Figure 5 ). alpha-Actin was used as a loading control. At least three independent experiments per protein were conducted and a representative result is presented. JP, junctophilin; CSQ, calsequestrin; Mg, mitsugumin; CaM, calmodulin. The amount of releasable Ca 2+ from the SR to the cytosol in response to TG (2.5 mu M ) ( b ) or resting cytosolic Ca 2+ level ( c ) was examined in myotubes treated with sildenafil, and histograms are shown for the normalized peak amplitude or area under the peak to the mean value of those from untreated controls as described in the 'Materials and methods' section. TG was applied to myotubes in the absence of extracellular Ca 2+ to avoid extracellular Ca 2+ entry. The results are presented as the means+-s.e. for the number of experiments presented in the parentheses of Table 2 . *, and the significant difference was compared with the untreated control ( P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Effect of the IT mutation on triadin and CSQ. ( a ) Representative western blots of triadin levels in soleus muscle from WT and IT mice at different ages. ( b ) Analysis of triadin levels. ( c ) RyR1 and CSQ localization in FDB fibres by immunocytochemistry. Magenta: RyR1, Green: CSQ. ( d , e ) Line scans of CSQ and RyR1 immunostaining in FDB fibres of IT ( e ) and WT ( d ) mice. ( f ) Analysis of the fluorescence profile of CSQ in WT and IT fibres, FWHM is full width at half maximum. Data are shown as mean+-s.e.m. * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 dCASQ- mice did not express any of the two CASQ isoforms. Western blot analysis of total homogenates prepared from hind limb (a), EDL, and Soleus (b and c) muscles showed that (i) in CASQ1- muscles CASQ1 was missing, whereas CASQ2 was still present, more in Soleus (slow twitch) and less in EDL (fast twitch); (ii) in dCASQ- muscles both isoforms were absent.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Association of syndecan-4 with calcineurin A (CnA). (A) Pull-down experiments with biotinylated peptides covering cytoplasmic domains of syndecan-1-4 were performed using recombinant His-trigger factor (TF)-CnA, and precipitates immunoblotted for presence of His-TF-CnA (n = 3). (B) Alignment of syndecan-4 and syndecan-2. Black boxes indicate similar amino acids within consensus region. Lysates from HEK-293 cells co-transfected with syndecan-4 and CnA subjected to immunoprecipitation with (C) anti-syndecan-4 (n = 3) or (D) anti-CnA (n = 3) were immunoblotted with anti-CnA or anti-syndecan-4, respectively. Pull-down experiments with biotinylated peptides covering non-phosphorylated or phosphorylated cytoplasmic part of syndecan-4 were performed using recombinant His-TF-CnA (E) or left ventricular (LV) tissue before immunoblotted for presence of endogenous CnA (n = 3) and PKC-alpha (n = 2) (F). (G) LV tissue extracts from WT-SHAB and WT-AB were immunoprecipitated with anti-syndecan-4 or control IgG and lysates and immunoprecipitates immunoblotted for presence of PKC-alpha, CnA and calmodulin (CaM, n = 2). (H) Relative pS179-syndecan-4/total syndecan-4 levels in aorta-banded (AB) versus sham-operated (SHAB) WT LV tissue (n = 3). Calsequestrin was used for loading control. Values are mean +- s.e.m.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1. IP 3 RII is up-regulated post-transcriptionally during hypertrophy in cardiac myocytes. (a) Quantification of the ratio of left ventricular weight to body weight in sham and AB adult rats. (b) Analysis of ANF mRNA expression by qPCR in sham and AB left ventricular samples. (c) Representative echocardiography of sham and AB rats. Dimensions indicate left ventricular diastolic dimension (LVDd) and posterior wall thickness (PWd), indices of ventricular function and hypertrophy, respectively. (d) Fractional shortening quantification (%) from echocardiographic analysis of eight sham and six AB rats. (e) Immunoblot for IP 3 RII in the membrane protein fraction of sham and AB ventricular lysates. Calnexin (CLNX) and calsequestrin (CSQ) are shown as loading controls. (f) Quantification of immunoblots as in e; n = 5. (g) Immunofluorescence image of ventricular myocytes isolated from sham and AB rats. IP 3 RII is shown in red, RyR2 in green, and DAPI in blue. Bar, 5.8 um. (h) Ratio of IP 3 RII to RyR2 quantified from immunofluorescence images as in g. For the sham condition, 16 cells from 2 independent preparations were analyzed. For the AB condition, 26 cells from 2 independent preparations were analyzed. (i) IP 3 RII mRNA expression measured by qPCR in sham/AB samples. (j) Nascent IP 3 RII RNA expression measured by qPCR in the sham/AB samples. Bar graphs are mean +- SEM from at least three independent experiments. *, P < 0.05; ***, P < 0.001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Validation of a few selected 2D DIGE proteins. (A), representative immunoblots of SLMAP, NDRG2, ERP29, and CRYAB expression levels in all experimental groups. Western blot of calsequestrin (CSQ) was used as a control of protein loading, and to normalize the densitometric data of each protein expression. (B-E), respective histograms of levels of SLMAP, NDRG2, ERP29, and CRYAB proteins (are expressed as fold) increases in TAC mouse groups (C and H) relative to their respective shams (S), in the FKBP12.6 overexpressing (K) group compared with wild-type mice (W) of both genders (M or F). Data are mean +- sem. * P < 0.05, TAC versus sham; $ P < 0.05, C versus. H; # P < 0.05, female versus male.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Confirmation of a direct PLM-NCX1 interaction ( A ) NCX1 and PLM were analysed in cytoplasmic and membrane fractions isolated from rat neonatal cardiomyocytes and LV using anti-NCX1 and anti-PLM antibodies. GAPDH and calsequestrin were used as controls for cytoplasmic and membrane fractions respectively. ( B ) Epitope mapping was performed by overlaying an array of immobilized overlapping 20-mer PLM peptides with anti-PLM (ab76597, left-hand panel). Amino acids in bold were relevant for anti-PLM binding. Immunoblotting without anti-PLM was used as a negative control (right-hand panel). ( C ) Rat LV, ( D ) brain or ( E and F ) lysate from HEK-293 cells co-transfected with NCX1 and PLM or PLM(S68D) was subjected to immunoprecipitation using anti-NCX1. Immunoprecipitates and lysate was immunoblotted with anti-NCX1 and anti-PLM antibodies. A specific anti-NCX1 blocking peptide and non-relevant rabbit IgG were used as negative controls. ( G ) Schematic presentation of biotinylated peptides covering PLM cyt and pSer 68 -PLM cyt (upper panel). The alpha-helical region is indicated. Immunoblotting analysis of the two biotinylated peptides using HRP-conjugated anti-biotin is shown in the lower panel. ( H ) Pull-down assay with biotin-PLM cyt and biotin-pSer 68 -PLM cyt against recombinant His-TF-NCX1 cyt (containing the cytoplasmic part of NCX1) using monoclonal anti-biotin-conjugated beads. Pull-down of NCX1 was analysed by immunoblotting using anti-NCX1. ( I ) Alignment of

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Transiently increased occurrence of longitudinal elements in the TAT system is accompanied by a persistent decrease in JPH2 post-MI and dissociation of Cav-3 into the cytosol. The occurrence of longitudinal elements of the TAT system transiently rises while JPH2 levels decrease and remain decreased from the earliest time point post-MI and Cav-3 levels increase and dissociate from the membrane 16-week post-MI. (A) Top panels: representative 40 x 10 um frames of the confocal images of cardiomyocytes at different time points (Scale bar equals 2 mum); Bottom panels: axial elements extracted mathematically from frames above as explained in 'Methods' section; (B) Number of longitudinal tubules; * P < 0.05 as calculated with mixed ANOVA followed by Wald chi 2 -test. Sample number N/n = 4/20 each; (C) Representative immunoblots for JPH2 and (D) of Cav-3 as well as the house keeping gene products GAPDH and CSQ, each sample was analysed in triplicates from 3 different animals (control = age matched animals); (E) Representative immunoblots showing dissociation of Cav-3 protein from membrane to cytosol at 4- and 16-week post-MI. SERCA2a was used as membrane fraction marker and GAPDH as cytosolic fraction marker. Ponceau staining was performed as overall loading control. (F) and (G) Density of respective JPH2 bands in (C) normalized to corresponding GAPDH bands, n = 3 each, and of Cav-3 bands in (D) normalized to CSQ bands. (H) Density of Cav-3 in cytosolic fractions of

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Casq mRNA in selected tissues from adult zebrafish (A) . The levels of RNA expression are relative to two different housekeeping genes, beta-2-microglobulin (B2M) and elongation factor1-alpha (EF1alpha). Mean values and standard error were determined from triplicate runs of the qPCR assay in three different tissue preparations. (B) . Anti-Casq antibodies reactivity on total homogenates (40 mug per lane) obtained from pooled (N = 3) skeletal muscle, heart, and brain of adult zebrafish. In the control lanes, (anti-rabbit) blot incubation was performed in the presence of anti-rabbit conjugate with alkaline phosphatase only. Molecular mass markers (in kDa) are indicated. (C) In vitro deglycosylation of Casqs. Immunodetection of Casq by CC antibody in membrane-enriched fraction P4 (see ""Materials and Methods"" section) after endoglycosidase F digestion, with 10 mug loaded per lane.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 Adult brain subcellular fractionation: immunochemical profile. Immunoblot analysis of brain subcellular fractions. Equal protein amounts (40 mug) from each fraction were analyzed with CC antibodies to recognize Casqs and with antibodies specific for synaptotagmin1 (SYT1), calreticulin, NDUFS3 (as described in ""Materials and Methods"" section). Raw data derived from densitometric analysis are presented in Supplementary Table S1 .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Immunofluorescence staining of sagittal brain sections area restricted to the cerebellum and the optic tectum (rostral to the right) decorated with CC (A,C-E) and MC (F,H-K) antibodies. (B,G) Images collected from control sections (see ""Results"" section) and acquired with the same conditions as in (A) and (F) , respectively. (A,B,F,G) Epi-fluorescence signals. (C-E,H-K) Images obtained by single optic section of confocal analysis. The box in (C) indicates the area shown at higher magnification in (E) . (D) PCL in a different region and focal plane. Arrows: axons of Purkinje cells. The abbreviations are the same as in Figure 4 . (A,C,F,H,I) Bar: 100 mum. (E) Bar: 25 mum. (D,J,K) Bar: 7.5 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 Co-staining of cerebellum with Casq and neuron-specific markers. (A-C) Epi-fluoresence signal obtained by double immunofluorescence with CC antibody (green) and anti-parvalbunin antibodies (red) in the cerebellum area. (C) A merge of the two images. Arrowheads indicate the axons of Purkinje cells. (D-F) Epi-fluoresence images obtained by double-staining with MC antibody (green) and anti-parvalbunin antibodies (red). (D) A merge of the two images. (G-I) Confocal images obtained by double-staining with MC antibody (green) and anti-NeuroD1 antibody (red). (J-L) Higher magnification of granule cells in the central area of the cerebellum double-labeled with MC (green) and anti-NeuroD1 antibody (red). Bar: 25 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Expression and phosphorylation of sarcoplasmic reticulum (SR) calcium-handling proteins in atria from JDP2-overexpressing mice. Original Western blots ( A , C ) and summarized data ( B , D ) from 8-9 atria isolated from mice overexpressing JDP2 for 5 weeks vs. WT mice ( p -values as indicated). Expression of SERCA2a, CSQ and RyR2 was analyzed, as well as phosphorylation of RyR2 at S2808. GAPDH was used as loading control for normalization. Note that the GAPDH signals shown in ( A ) for SERCA2a and in ( C ) for pS2808 are identical, as the proteins were derived from the same Western blot.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 2 (a) Representative immunoblot of the collagenase matrix metalloproteinase-1 at 57/52 kDa (proMMP-1, latent) and active MMP-1 at 46 kDa. MMP-1 control = MMP-1 protein control (Sigma-Aldrich, Deisenhofen, Germany), WT = wild type, TGF-beta1- TG = Alb/TGF-beta1 (Cys223,225Ser) transgenic mice at 8 weeks of age, MET = Metoprolol, TEL = Telmisartan, TGF-beta1-sR-Ab = soluble TGF-beta1 receptor antibody (upper panel). Western blot data were normalized to calsequestrin as a non-regulated protein. Densitometric quantification of the active MMP-1 protein expression is shown (lower panel). Data are represented as mean +- SEM; n = 5 per group. (b) Representative immunoblot of matrix metalloproteinase-13 (MMP-13) protein expression at 48 kDa (active form). WT = wild type, TGF-beta1-TG = Alb/TGF-beta1 (Cys223,225Ser) transgenic mice at 8 weeks of age, MET = Metoprolol, TEL = Telmisartan, TGF-beta1-sR-Ab = soluble TGF-beta1 receptor antibody (upper panel). Western blot data were normalized to calsequestrin as a non-regulated protein. Densitometric quantification of the active collagenase MMP-13 is shown (lower panel). Data are represented as mean +- SEM; n = 5 per group.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 3 (a) Representative immunoblot of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) protein expression at 28 kDa. TIMP-1-control = TIMP-1 protein control (Sigma-Aldrich, Deisenhofen, Germany), WT = wild type, TGF-beta1-TG = Alb/TGF-beta1 (Cys223,225Ser) transgenic mice at 8 weeks of age, MET = Metoprolol, TEL = Telmisartan, TGF-beta1-sR-Ab = soluble TGF-beta1 receptor antibody (upper panel). Western blot data were normalized to calsequestrin as a non-regulated protein. Densitometric quantification of the collagenase inhibitor TIMP-1 is shown (lower panel). Data are represented as mean +- SEM; n = 5 per group. (b) Representative immunoblot of tissue inhibitor of matrix metalloproteinase-4 (TIMP-4) protein expression at 29 kDa. WT = wild type, TGF-beta1-TG = Alb/TGF-beta1 (Cys223,225Ser) transgenic mice at 8 weeks of age, MET = Metoprolol, TEL = Telmisartan, TGF-beta1-sR-Ab = soluble TGF-beta1 receptor antibody (upper panel). Western blot data were normalized to calsequestrin as a non-regulated protein. Densitometric quantification of the MMP-inhibitor TIMP-4 is shown (lower panel). Data are represented as mean +- SEM; n = 5 per group.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 2 A Immunoblot analysis of MMP-13 collagenase protein expression and densitometric quantification. Corresponding blot of the housekeeping protein calsequestrin is shown below. B Densitometric quantification of the collagenase pro-MMP-1 (latent) and active MMP-1 protein expression measured by immunoblotting

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 4 Immunoblot and densitometric quantification of tissue inhibitor of matrix metalloproteinase-2 (TIMP-2; 24 kD). TIMP-2 expression continuously increased in beta1TG mice with progressive interstitial fibrosis and ventricular dysfunction. Corresponding blot of the housekeeping protein calsequestrin is shown below; n=5/group

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 5 A Immunoblot and densitometric quantification of membrane-type-1 MMP protein (58 kD) expression of beta1TG mice and WT. Corresponding blot of the housekeeping protein calsequestrin is shown below; n=5/group. B Quantitative real-time PCR of MT1-MMP mRNA. Ct values are presented as a ratio of MT1-MMP mRNA to the internal standard GAPDH mRNA

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 6 Immunoblot and densitometric quantification of integrin-linked kinase (ILK) protein expression of beta1TG and age matched WT. ILK is recognized at 59 kD (ILK control) and significantly downregulated in mice with severe interstitial fibrosis and ventricular dilatation at 12 months of age. Corresponding blot of the housekeeping protein calsequestrin is shown below

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 Transient outward K + channel expression in transgenic atrial myocytes. Experimental animals involved wild-type mice (WT) and transgenic mice (TG) expressing the repressor isoform of cyclic adenosine monophosphate response element modulator, CREM-IbDeltaC-X, and were distributed according to age in 6-week-old (WT 6w and TG 6w ) and 12-week-old (WT 12w and TG 12w ) groups. A , Representative transient outward K + current (I to ) traces recorded as -40 mV sensitive currents from atrial cardiomyocytes of WT 12w and TG 12w . For clarity, current density (expressed in picoamperes/picofarads [pA/pF]) traces from -40 mV with +20 mV increments are displayed. B , Mean+-SE of current-voltage plots of the peak current density (I to ). For WT 6w , n/N are 41/15, for TG 6w n/N are 22/14, for WT 12w n/N are 35/19, and for TG 12w n/N are 20/16. Ca through d , Representative immunoblots and quantification of protein expression of calsequestrin (CSQ; Ca ) normalized to Ponceau and of K + voltage-gated channel subfamily D member 2 (Kv4.2, Cb ) and 3 (Kv4.3, Cc ), and K + channel interacting protein 2 (KChIP2) ( Cd ) normalized to CSQ. Data show mean+-SE of N=7 mice per group, except for Kv4.3, where N=6 as the result of an undetectable band in TG 12w . * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Inward rectifier K + current I K1 expression and function in transgenic atrial myocytes. Experimental animals involved wild-type mice (WT) and transgenic mice (TG) expressing the repressor isoform of cyclic adenosine monophosphate response element modulator, CREM-IbDeltaC-X, and were distributed according to age in 6-week-old (WT 6w and TG 6w ) and 12-week-old (WT 12w and TG 12w ) groups. A , Representative traces of inward rectifiers K + channel (I K1 ) recorded in WT 12w and TG 12w myocytes before and after application of 10 mumol/L BaCl 2 using a ramp protocol (inset, scale bar 0.2 seconds). The traces show current densitiy (expressed in picoamperes/picofarads [pA/pF]) over time. ( Aa ). Current-voltage plots of I K1 averaged from traces of all cells. For WT 6w , n/N are 8/5, for TG 6w n/N are 10/6, for WT 12w n/N are 13/7, and for TG 12w n/N are 9/6 ( Ab ). B , Data show mean+-SE of relative mRNA levels of atrial K + voltage-gated channel subfamily J member 2 ( Kcnj2 ) and 4 ( Kcnj4 ) normalized to WT 6w vs Hprt1 (hypoxanthine phosphoribosyltransferase 1) as the housekeeping gene; N=8 per group. Y-axes scale is Log2. Ca through c , Representative immunoblots ( Ca ) and quantification of K + inwardly rectifying channel subunits Kir2.1 ( Cb ) and Kir2.3 ( Cc ) protein levels normalized to calsequestrin (CSQ). Data show mean+-SE of 7 mice per group. Da through b , Representative action potentials recorded before (normal Tyrode [NT] as control) and after application

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Acetylcholine-activated K + current (I KACh ) expression and function in transgenic atrial myocytes. Experimental animals involved wild-type mice (WT) and transgenic mice (TG) expressing the repressor isoform of cyclic adenosine monophosphate response element modulator, CREM-IbDeltaC-X, and were distributed according to age in 6-week-old (WT 6w and TG 6w ) and 12-week-old (WT 12w and TG 12w ) groups. A , Representative traces recorded before and after application of 10 mumol/L acetylcholine using the ramp protocol (inset, scale bar 0.2 seconds) in 1 isolated WT 12w atrial cardiomyocyte. The acetylcholine-sensitive current derived by subtraction is I KACh . The traces show current densitiy (expressed in picoamperes/picofarads [pA/pF]) over time. ( Aa ). Current-voltage plots of I KACh averaged from traces of all cells. n/N for WT 6w are 12/6, for TG 6w are 13/7, for WT 12w are 12/6, and for TG 12w are 10/6 ( Ab ). B , Relative mRNA levels of K + voltage-gated channel subfamily J members 3 ( Kcnj3 ) and 5 ( Kcnj5 ) measured in atrial tissue, with Hprt1 (hypoxanthine phosphoribosyltransferase 1) as the housekeeping gene. Data were normalized to WT 6w . Y-axes scale is Log2. Data show mean+-SE of 8 per group. Ca through c , Representative immunoblots ( Ca ) and quantification of G protein-coupled inwardly rectifying K + channel alpha subunits 1 (GIRK1, Cb ) and 4 (GIRK4, Cc ) protein levels normalized to calsequestrin (CSQ). Data show mean+-SE of N=7 mice per group. Da t

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2. A, Exemplificative ventricular sections (hematoxylin-eosin stain) of CASQ2 WT/WT , CASQ2 R33Q/WT , and CASQ2 R33Q/R33Q (100x). Bars&equals10 &mgrm. B, Ventricular cardiomyocytes from CASQ2 WT/WT and CASQ2 R33Q/R33Q immunostained with rabbit anti-CASQ and mouse anti-alpha-actinin antibodies. Bars&equals20 &mgrm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2. miR-133a regulates IP 3 RII expression. (a) Conservation of the miR-133a seed sequence between the human, mouse, and rat Itpr2 3'UTR. (b) miR-133a expression in sham and AB samples measured by qPCR. (c) miR-1 expression in sham and AB samples. (d) Schematic representation of the luciferase reporter construct used to measure miR-133a repressive activity at the Itpr2 3'UTR. (e) Luciferase activity in HEK293 cells transfected with miR-133a and either the Itpr2 3'UTR reporter or mut- Itpr2 3'UTR reporter. (f) Immunoblot for IP 3 RII in left ventricular samples from wild-type (WT) or miR-133a-1, miR-133a-2 double knockout mice (dKO). (g) Quantification of immunoblots for IP 3 RI/II/III from WT/dKO samples as in f; n = 6. (h) IP 3 RI/II/III mRNA expression in samples as in f. Bar graphs represent the mean +- SEM from at least three independent experiments. *, P < 0.05; **, P < 0.01.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4. Antagonism of miR-133a-mediated regulation of IP 3 RII induces the hypertrophic response. (a) Immunoblot for IP 3 RII and SRF in NRVMs transfected with control or IP 3 RII-specific target protector (TP) oligos. alpha-Actinin is shown as a loading control. (b) ANF protein expression in NRVMs transfected as in a. (c) Cell surface area measurements of NRVMs transfected as in panel a. More than 500 cells were measured per condition. (d) Quantification of the fold change in the number of calcium release events elicited by IP 3 -ester in ARVMs infected with control or miR-133a adenovirus. For each condition, at least eight ARVMs were analyzed from three separate experiments. (e) IP 3 RII protein, mRNA, and nascent RNA expression in WKY and SHR myocytes measured by immunoblotting or qPCR. (f) miR-133a expression analyzed by qPCR in WKY/SHR myocytes. (g) Quantification of the hypertrophic phenotype by measurement of ANF mRNA expression in GFP/miR-133a-GFP overexpressing WKY and SHR ventricular myocytes. (h) Measurement of LVPWd by echocardiography in mice at baseline and after infusion with saline or miR-133a antagomir for 1 mo. (i) Immunoblot for IP 3 RII in left ventricular samples from mice treated with saline/antagomir for 1 mo. (j) Quantification of immunoblots as in i; n = 3. Bar graphs are mean +- SEM from at least three independent experiments. *, P < 0.05; ***, P < 0.001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6. Manipulation of IICR in vivo alters miR-133a expression. (a) Immunoblot for IP 3 RII in left ventricular lysates from IP 3 RII transgenic mice. Tetracycline transactivator protein (tTA) mice are used as controls. (b) miR-133a expression measured by qPCR in tTA/IP 3 RII transgenic mice subjected to sham or TAC procedures. For the tTA sham/TAC and IP 3 RII-TAC conditions, three mice were analyzed. For the IP 3 RII-sham condition, two mice were analyzed. (c) Representative immunoblot for IP 3 RII in tTA ventricular lysates +- TAC. (d) qPCR analysis of miR-133a expression in ventricular lysates from tTA controls and IP 3 -sponge (sponge) expressing mice subjected to sham or TAC procedures. For the tTA sham/TAC and IP 3 -sponge-TAC conditions, three mice were analyzed. For the sponge-sham condition, two mice were analyzed. (e) Quantification of immunoblots for IP 3 RII from sham or TAC operated tTA and sponge mice. Two mice were analyzed for the tTA-sham and sponge-sham conditions. Three mice were analyzed for the tTA-TAC and sponge-TAC conditions. (f) qPCR measurement of IP 3 RII mRNA and nascent RNA expression in tTA and sponge mice. Three mice were analyzed for the tTA condition, two mice were analyzed for the sponge condition. (g) Example immunoblot of ventricular lysates from wild-type (WT) and IP 3 RII knockout (KO) mice. (h) miR-133a expression measured using qPCR in four WT and four KO mouse samples. Bar graphs show mean +- SEM. *, P < 0.05; **, P < 0.01.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 7. IICR affects miR-133a expression through modulation of SRF activity. (a) Luciferase reporter assay of SRF transcriptional activity in GFP/GFP-5P overexpressing NRVMs cultured in the presence and absence of 100 nM ET-1 for 24 h. (b) Immunoblot for SRF in control and ET-1-treated NRVM lysates. (c) Quantification of immunoblots as in b; n = 6. (d) Immunoblot for SRF in control or SRF-overexpressing NRVMs. (e) Immunoblot for SRF in control or siSRF-transfected NRVMs. (f) qPCR analysis of miR-133a expression in GFP control, SRF overexpressing, or SRF knockdown NRVMs. (g) Immunoblot to show IP 3 RII expression in GFP-control, SRF-overexpressing, and SRF knockdown NRVMs. (h) Quantification of immunoblots as in g; n = 4. (i) IP 3 RII nascent RNA expression in SRF-overexpressing NRVMs measured using qPCR. (j) qPCR analysis of miR-133a expression in control and DN-SRF-overexpressing NRVMs treated with ET-1. (k) miR-133a expression in control or HOP-overexpressing NRVMs cultured in the presence and absence of 100 nM ET-1 for 24 h. (l) Cell surface area quantification in control/HOP-overexpressing NRVMs. More than 300 cells taken from 4 separate experiments were measured per condition. (m) qPCR analysis of miR-133a expression in NRVMs treated with ET-1 in the presence and absence of 0.4 uM trichostatin A (TsA). (n) qPCR analysis of ANF expression in NRVMs treated as in k. Data are mean +- SEM from at least three independent experiments.*, P < 0.05; **, P < 0.01.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Remodeling of proteins involved in calcium signaling pathways in the PKP2-Hz mouse. Representative western blots (left) and average densitometry (right; n = 5 for all groups) of ankyrinB (AnkB), calsequestrin-2 (Casq2), Ca v 1.2, and SERCA2a, measured from wildtype and PKP2-Hz ventricular lysates of three-month-old mice. Left upper panels represent western blots, bottom panels according to ponceau staining used for quantification (mean +- SEM, * p < 0.05).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Pro-arrhythmic cardiac remodeling in PKP2-Hz mice exposed to exercise training. ( A ) Representative pictures of Picrosirius red staining (left panel) in the inner myocardium of wildtype and PKP2-Hz murine heart sections, of mice exposed to exercise training; scale bar, 500 um. Quantification of collagen abundance (right panel) in overview slides of whole heart sections (WT; n = 5 and PKP2-Hz; n = 5, mean +- SEM). ( B ) Hematoxylin and eosin staining of wildtype and PKP2-Hz heart sections, of mice exposed to exercise; scale bar, 100 um. ( C ) Quantification of heart weight (HW) to body weight (BW) ratio in both groups (WT; n = 5 and PKP2-Hz; n = 5, mean +- SEM). ( D ) Representative western blots (left panel) and average densitometry (right panels, n = 5 for all groups) of AnkyrinB (ANKB), Ca v 1.2, and calsequestrin-2 (Casq2) measured from wildtype (WT) and PKP2-Hz ventricular lysates of mice exposed to exercise training. Left upper panels represent western blots, bottom panels according ponceau staining used for quantification (mean +- SEM, * p < 0.05). ( E ) Immunofluorescence staining for Cx43 (red) and Ncad (green) in right ventricular heart sections of WT and PKP2-Hz mice; scale bar, 100 um. Upper right panel shows magnified shots of according merged pictures, indicated by the white boxes. Lateral Cx43 expression is more pronounced in PKP2-Hz hearts exposed to exercise training, indicated by white arrows. ( F ) Left ventricular fractional shortening examined vi