MA3-913

antibody from Invitrogen Antibodies

Targeting: CASQ1 CASQ, PDIB1

Provider product page for MA3-913

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [81]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [3]
Immunohistochemistry [2]
Other assay [26]

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Product number: MA3-913 - Provider product page
Provider: Invitrogen Antibodies
Product name: Calsequestrin Monoclonal Antibody (VIIID12)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MA3-913 detects calsequestrin from human, mouse, rat, canine, porcine, chicken, and rabbit skeletal muscle tissues. This antibody recognizes calsequestrin in both type I (slow) and type II (fast) skeletal muscle tissues.
Antibody clone number: VIIID12
Concentration: Conc. Not Determined

CASQ1 protein structure - MA3-913 shown in red.

Supportive validation

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Experimental details: Immunofluorescent analysis of Calsequestrin using Anti-Calsequestrin Monoclonal Antibody (VIIID12) (Product # MA3-913) shows staining in Hela Cells. Calsequestrin staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Calsequestrin (Product # MA3-913) at a dilution of 1:100 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

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Experimental details: Immunofluorescent analysis of Calsequestrin using Anti-Calsequestrin Monoclonal Antibody (VIIID12) (Product # MA3-913) shows staining in MCF-7 Cells. Calsequestrin staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Calsequestrin (Product # MA3-913) at a dilution of 1:100 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Calsequestrin using Anti-Calsequestrin Monoclonal Antibody (VIIID12) (Product # MA3-913) shows staining in U251 Cells. Calsequestrin staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Calsequestrin (Product # MA3-913) at a dilution of 1:100 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

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Experimental details: Immunohistochemistry was performed on normal deparaffinized Human heart tissue tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing Calsequestrin (Product # MA3-913) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on normal deparaffinized Human skeletal muscle tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a mouse monoclonal antibody recognizing Calsequestrin (Product # MA3-913) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

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Experimental details: Fig 2 PLN phosphorylation and expression levels of other Ca 2+ regulatory proteins. (A) Western blot analyses demonstrating that monomeric p-PLN/PLN ratio is significantly lower in both Pln OE and Pln OE / Sln KO soleus muscles compared with WT (n = 5-6 per genotype). Western blotting for other major Ca 2+ regulatory proteins SERCA1a (B, 110 kDa), SERCA2a (C, 110 kDa), RyR (D, 565 kDa), DHPR (E, 170 kDa), and CSQ (F, 63 kDa) did not reveal any significant differences across genotypes (n = 5-8 per genotype, with a 6 mug load for all proteins). *Significantly different from WT using a a one-way ANOVA with a Tukey's post-hoc, p

Supportive validation

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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

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Experimental details: Immunofluorescence experiments on cross sections of biopsies from patients 3 and 4 revealed that nearly all tubular aggregates contained CASQ1. The vast majority of CASQ1 positive tubular aggregates were also positive for STIM1 and RYR1, whereas ORAI1 was rarely detected and at much lower extent in CASQ1 positive aggregates. Bars: 20 mum

Supportive validation

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Experimental details: 2 Protein expression in embryonic chicken cardiac myocytes Embryonic chicken cardiac myocytes were infected with Ad.Sr1 (expressing SERCA1 ) or Ad.Sr2a (expressing SERCA2a ), and cell lysates were probed with various monoclonal antibodies. A , right, antibody to the c-myc tag found only in exogenous SERCA1 and SERCA2a; middle, antibody to SERCA1; left, antibody to endogenous and exogenous SERCA2a. Lane headings: C, control; Sr1, Ad.Sr1 infected; Sr2a, Ad.Sr2a infected. B , antibody to phospholamban. C , antibody to calsequestrin. For B and C , each lane was run in duplicate.

Supportive validation

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Experimental details: 5 Figure Abundance of key calcium-release proteins in the diaphragm does not change with NaNO 3 supplementation in old mice A , representative membrane immunoblot (top) and total protein gel (bottom) images. Quantification of immunoblot signal normalized to total protein for calsequestrin (CSQ) ( B ), DHPR (Ca v 1.1) ( C ), and ryanodine receptor 1 (RyR1) ( D ). Bars represent mean value and symbols are individual data from n = 6 (control) and 7-8 (NaNO 3 ) mice per group. P values are from unpaired Student's t tests.

Supportive validation

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Experimental details: Figure 2. Protein content in patients' microsomes. ( A ) Western blot analysis of glycogen phosphorylase (GP) for 25 subjects, in a luminescence scale with black as 0 and white as saturating value. The numbers above each lane are patient identifiers that apply to all 25-lane gels shown in the article. The twelve lanes under the black bar have protein from MHN patients; those under the red bar are from MHS patients, except #144, which was reclassified as MHN. ( B ) Ponceau-stained gel that originated all blots in the figure. A and B illustrate our custom quantitative analysis. The content of every protein quantified in blots was calculated as the signal mass within region a above background (average level in b). Content was normalized for quantity of preparation in the lane dividing by the signal similarly calculated in the gel in B. The signal in B is computed in a large area of the lane to average multiple proteins in the fraction. The method is fully demonstrated in Video 1 . Arrows in B mark two bands, near 100 and 180 kDa, with a visibly greater signal in the MHS group. Their main components were GP and glycogen debranching enzyme (GDE) respectively. ( C-E ) Western blots of GDE, calsequestrin 1 and FKBP12 in different sections of gel B (identified by molecular weight markers at left). ( F and G ) Box plots of GP and GDE content. In both cases the content was greater on average for MHS and the differences significant (data in Table 1 ). ( H ) GP vs. GDE in blots A and C;

Supportive validation

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Experimental details: Figure 3. Phosphorylation of glycogen phosphorylase. ( A, B ) Western blots of 13 MHN and 12 MHS whole tissue lysates, identified in Figure 2 . Blots were derived from different gels loaded with aliquots of the same preparations. Originating gels for this blot and others are in Figure 3--figure supplement 1 . ( C ) The distribution of band signals in blots A and B. Statistical measures are listed in Table 1 . GP ( all-forms ) and GP a were higher in the MHS (p=0.07 for GP and 3 10 -4 for GP a ). ( D ) The median content ratio GP a /GP was 42% higher in the MHS (p = 0.005). ( E ) Cross-plot demonstrating correlation between GP a content in whole muscle and GP in microsomes ( r = 0.64, p = 5 10 -4 ). ( F ) Western blot of GP a in the microsomal fractions studied in Figure 2 . ( G, H ) Distributions of GP a in blot F and of the content ratio GP a /GP (the all-forms GP content was determined with blot A of Figure 2 ). GP a was higher by 71% (p = 0.05) but the phosphorylation ratio did not change significantly in the microsomal fraction (p = 0.28). ( I ) GP a vs. GP in microsomes. The correlation is high and highly significant ( r = 0.72, p < 10 -4 .). Figure 3--figure supplement 1. Protein gels for blots. ( A, B ) Ponceau-stained whole tissue extract gels used to derive Western blots of PhK, GPa and GDE ( A ) and all-forms GP ( B ). C-F. Ponceau-stained microsomal extract gels used to derive Western blots of GS ( C ), GSa ( D ), GDE ( E ) and GLUT4 and Casq1 ( F ). The signal in

Supportive validation

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Experimental details: Figure 5. Phosphorylated glycogen phosphorylase, GP a , localizes at SR. ( A ) Sections of a z -stack (corrected as in Figure 4 ) of human muscle co-stained for GP a and Casq1(I.D. #164, MHN). ( B ) 3-D renderings of the z -stacks that contained the sections in A. Both proteins delineate two terminal cisternae, TC, in every triad. The inset in Ac shows close proximity of the imaged proteins, without actual colocalization (documented with Figure 5--figure supplement 1 ). ( C, D ) Sections and stack from a human myofiber co-stained for GP a and STIM1 (I.D. #164, MHN). GP a delineates two TC in every triad (Ca, Da). STIM1 forms clusters in close proximity to the GP a bands, without actual colocalization. Minor quantities of both proteins are located outside triads. A large quantity of STIM1, unaccompanied by GP a , appears inside a nucleus (cf. (). These images are the first to resolve TC with conventional optics (a resolution of ~100 nm is estimated with Figure 5--figure supplement 2 ). Figure 5--figure supplement 1. Analysis of colocalization of GP a and STIM1. ( A ) Enlarged part of text Figure 5 panel Cc. ( B ) Object-based analysis, by JACoP, of images in panels Ca (GP a ) and Cb (STIM1) of Figure 5 . Centers of signal mass of STIM1 'objects' (i.e. automatically detected connected areas of fluorescence) are marked by a green symbol. Centers of objects in the GP a image are marked in red. Green and red locations are different but close. ( C ) Van Steensel's approach to calcu

Supportive validation

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Experimental details: Figure 4 Limited SIT-induced changes in mRNA and protein expression of other SR Ca 2+ -handling proteins than RyR1. ( a ) Mean and individual data of the ratio of mRNA expression 1 h after to that before the first (Untrained) and last (Trained) SIT sessions in individuals treated with vitamins ( n = 9) or with placebo ( n = 7); dashed line indicates no difference between before and after exercise. Two-way repeated measures ANOVA showed a general group difference ( p < 0.01) in the untrained state. Difference between vitamins and placebo groups in relative mRNA expression of individual genes: * p < 0.05, *** p < 0.001. ( b ) Representative original Western blots from one individual treated with vitamins (Vit) and one treated with placebo (Pla). Biopsies taken before the first (untrained, U) and the last (trained, T) SIT session. Lower panels show stain-free loading controls. ( c ) Mean and individual data of the relative protein expression in biopsies obtained in the untrained and the trained state in individuals treated with vitamins (n = 10) or with placebo ( n = 6-7). Data expressed as the ratio of the expression in the trained to that in the untrained state; dashed line indicate no difference between the two states. No significant differences between the untrained and the trained state or between vitamin and placebo-treated individuals were observed with two-way repeated measures ANOVA. Data are shown as mean +- SD; vitamin group, black bars; placebo group, white bars.

Supportive validation

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Experimental details: Figure 2 ACTN3 deficiency is accompanied by a shift toward a slower skeletal muscle phenotype (A-D) Summary data (mean +- SEM) and representative western blots of the SR Ca 2+ -handling proteins SERCA1, SERCA2a, CSQ, and CSQ2 in muscle of RR (n = 8) and XX (n = 7) individuals. Band intensities were normalized to their respective myosin loading controls. Data expressed relative to the mean value of the RR group, which was set to 1.0. Statistical difference between the two groups was assessed with unpaired t test. (E) Silver-stained gels were used to assess the distribution of MyHC isoforms in RR (n = 7) and XX (n = 7) individuals. Right part shows a representative example of the distribution of MyHC in RR and XX individuals. The total staining of the three MyHC bands was set to 1 in each subject. Statistical difference between the two groups was tested with unpaired t test. (F) Mean-centered sigma-normalized heatmap of differentially expressed proteins (p < 0.05). (G) Volcano plot of all identified proteins (n = 601) expressed as fold-change (FC) in XX compared to RR individuals. Differentially abundant proteins are indicated in red.

Supportive validation

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Experimental details: Figure 5 Expression levels of muscle calcium-regulated proteins in mice after a high-fat diet. Protein levels in control mice ( CONT ) and high-fat diet-fed mice ( HFD ; upper panel: 4 weeks; lower panels: 12 weeks). Left: representative western blots of RyR, DHPR , CSQ , SERCA 1 and PV proteins (A: 4 weeks; C: 12 weeks). A protein expression levels expressed relative to the value of CONT mice (B: 4 weeks; D: 12 weeks). Values are means +- SE ( n = 7).

Supportive validation

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Experimental details: Figure 7 Variations of gene and protein expression involved in Ca 2+ handling in response to 10-day bed rest Calsequestrin1-2, SERCA1, SERCA2 and TOM20 amount in muscle homogenates as obtained by western blot at baseline (BR0) and after 10 days of bed rest (BR10) ( A ). Representative western blot of CASQ1-2, SERCA1, SERCA2 and TOM20 ( B ). CASQ1 and CASQ2 gene expression (encoding for Calsequestrin1 and Calsequestrin2, respectively) ( C , D ), ATP2A1 and ATP2A2 gene expression (encoding for SERCA1 and SERCA2, respectively) ( E , F ); RYR1 gene expression (encoding for Ryanodine Receptor 1) ( G ). All RNA transcripts are reported as normalized read count at BR0, after 5 days of bed rest (BR5) and at BR10. Results shown as means +- SD, individual data represented as scatter plots. * P < 0.05 BR10 vs BR0, ** P < 0.01 BR10 vs BR0, **** P < 0.0001 BR10 vs BR0.

Supportive validation

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Experimental details: Figure 1 Relative levels of CRU proteins in crude homogenates from skeletal muscle. Identical amounts (25 ug/lane) of microsomal fraction of skeletal muscles from WT, Tdn-, Jct- and Tdn-/Jct mice were loaded and immunoblotted with several antibodies. Membranes were tested for expression of triadin (Tdn), junctin (Jct), ryanodine receptor (RyR1), Dihydropyridine receptors (Cav1.1), Calsequestrin (CASQ), SERCA-1 pump (SERCA), FK506 binding protein (FKBP12), Junctophilin-1 (JP-1) and Histidin-rich Ca 2+ binding protein (HRC) as described in Material and Methods. Band intensity for each protein was normalized to GAPDH expression to correct for loading and plotted as fraction of its WT counterpart (dotted line). Data presented as mean +- SD of 3-7 independent blots. * p