MA3-922

antibody from Invitrogen Antibodies

Targeting: PLN CMD1P, PLB

Provider product page for MA3-922

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [176]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [1]
Immunohistochemistry [4]
Other assay [47]

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Product number: MA3-922 - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospholamban Monoclonal Antibody (2D12)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: MA3-922 detects phospholamban (PLB) in rabbit, human, pig, sheep, chicken, canine, guinea porcine, rat, and mouse. MA3-922 has been successfully used in Western blot, immunofluorescence, immunohistochemistry and immunoprecipitation procedures. By Western blot, this antibody detects a 25 kDa protein under non-reducing conditions representing the pentameric form of PLB and a 5 kDa protein under reducing conditions representing the monomeric form of PLB in canine cardiac extracts. Immunofluorescence staining of PLB in rat papillary muscle tissue with MA3-922 results in diffuse staining of the network sarcoplasmic reticulum (SR). MA3-922 can also be used to increase the calcium uptake in ventricular SR vesicles analogous to the phosphorylation of PLB following adrenergic stimulation. This antibody is recommended for immunofluorescent staining of tissues. The MA3-922 immunogen is a synthetic peptide corresponding to residues D(2) K V Q Y L T R S A I R R A S T I E M P Q Q A R(25) of canine PLB. This antibody recognizes an epitope between amino acid residues 9-17 of canine PLB.
Reactivity: Human, Mouse, Rat, Canine, Chicken/Avian, Guinea Pig, Porcine, Rabbit
Host: Mouse
Isotype: IgG
Antibody clone number: 2D12
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

PLN protein structure - MA3-922 shown in red.

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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 Phospholamban (Product # MA3-922) 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.

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Experimental details: Immunofluorescent analysis of Phospholamban (green) showing staining in the cytoplasm of human heart tissue (right) compared to a negative control without primary antibody (left). Formalin-fixed tissue was 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. Tissue was probed with a Phospholamban monoclonal antibody (Product # MA3-922) in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4ºC in a humidified chamber. Tissue was washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. Nuclei were stained with Hoechst or DAPI (blue). Images were taken at a magnification of 60x.

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Experimental details: Immunofluorescent analysis of Phospholamban (green) showing staining in the cytoplasm of mouse heart tissue (right) compared to a negative control without primary antibody (left). Formalin-fixed tissue was 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. Tissue was probed with a Phospholamban monoclonal antibody (Product # MA3-922) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4ºC in a humidified chamber. Tissue was washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. Nuclei were stained with Hoechst or DAPI (blue). Images were taken at a magnification of 60x.

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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 1:200 with a mouse monoclonal antibody recognizing Phospholamban (Product # MA3-922) 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.

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Experimental details: Figure 4 Calcium Handling Properties of hiPSC-derived cardiomyocytes under High Glucose Condition and Empagliflozin. ( A ) Representative tracings of whole-cell calcium transients in hiPSC-derived cardiomyocytes; (B) Amplitudes of whole-cell calcium transients; (C) Maximal upstroke velocity of whole-cell calcium transients; and (D) Maximal decay velocity of whole-cell calcium transients. Abbreviations: NG: normal glucose; HG: high glucose; HG + E: high glucose and empagliflozin; and HG, HG + E: high glucose for 7 days followed with empagliflozin. (E) The expression of ATP2A2 , RYR2 and NCX1 was evaluated by quantitative PCR analysis using the expression of TNNT2 as an internal control. Abbreviations: NG: normal glucose; HG: high glucose; HG + E: high glucose and empagliflozin; and HG, HG + E: high glucose for 7 days followed with empagliflozin. (F) Protein level of phospho-phospholamban and total phospholamban was evaluated by Western blot analysis, the protein level of beta-actin was used as internal reference. * p < 0.05 and ** p < 0.01. For the full-length image of gels and blots shown in this figure, please refer to Supplementary Figs S4 and S5 .

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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

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Experimental details: Figure 2--figure supplement 3. DWORF binding to SERCA2a displaces PLN in a dose-dependent manner and enhances SERCA activity. ( A ) Immunoprecipitations of Myc-SERCA2a were performed on lysates of HEK293 cells co-transfected with equal amounts of HA-PLN and Myc-SERCA2a (1 ug) and increasing amounts of HA-DWORF. Western blots on input samples (bottom) and bound immunoprecipitated fractions (top) reveal that DWORF binding to SERCA2a competitively displaces PLN. ( B, C ) Ca 2+ -dependent Ca 2+ -uptake assays were performed using homogenates from HEK293 cells co-transfected with equal amounts of SERCA2a and PLN (1 ug) and increasing amounts of DWORF. Co-transfection of cells with PLN (dark grey line) results in a rightward shift of the Ca 2+ -affinity curve as compared to control cells (SERCA2a alone, black) ( B ). Co-transfection with increasing levels of DWORF relieves the inhibitory effect of PLN on SERCA2a in a dose-dependent manner as evidenced by a leftward shift of the affinity curve back toward control values. Cells expressing SERCA2a and DWORF in the absence of PLN do not exhibit enhanced SERCA activity (purple), indicating that DWORF exerts its' stimulatory effect on SERCA through the displacement of PLN. ( C ) Mean K Ca values from n = 4 separate experiments are represented as bar graphs (+-SD). p-value *p

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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.

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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

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Experimental details: Fig 6 Immunoblotting. Immunoblot analysis of key Ca 2+ handling proteins and phosphorylation was performed on tissue from left ventricles. SERCA2A abundance (A). PLB abundance (B) and phosphorylation on SER16 (C) and THR17 (D). NCX abundance (E). RyR abundance (F) with phosphorylation on SER2808 (G) and SER2814 (H). CaMKII abundance (I) and CaMKII phosphorylation on THR286 (J). PP1 (K) and PP2A (L) abundance. n heart = 6 for all analysis. *p

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Experimental details: Fig 5 Densitometric analysis of Western blots. (A) SERCA2a; (B) phospholamban (PLB), (C) PLB to SERCA2a ratio, in hearts from male and female sham and gonadectomized rats. (SM: sham male; SF: sham female; GM: gonadectomized male; GF: gonadectomized female). The values are expressed as the means +-S.E.M (n = 6 per group). *P < 0.05 vs. females of the same group; + P < 0.05 vs. sham animals of the same sex (two-way ANOVA and Fischer test).

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Experimental details: Figure 5 Expressions of PLB (A), pPLB-ser16 (B), PKA (C) and PP-1 (D)normalyzed by beta-actin. PKA: protein kinase A; PLB:dephosphorylated phospholamban; pPLB-ser16: phosphorylatedphospholamban in serin-16; PP-1: phosphatase-1. Control (n = 6) andobese (n = 6). Data are expressed as mean +- standard-deviation.Student's t-test * p

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Experimental details: Fig 8 TRPC4beta attenuates hypertrophic growth in cardiomyocytes after GPCR stimulation. A, Hypertrophic growth of cardiomyocytes was measured as an increase of the cell surface after angiotensin II/phenylephrine (Ang II/PE) or vehicle (CON) treatment for 24 h. Red: alpha-actinin; Blue: DAPI. * P

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Experimental details: Fig 4 Alterations of sarcoplasmic reticulum Ca 2+ -ATPase (Serca) 2a and its regulator phospholamban (PLN) during AF. Total PLN ( a ) and PLN phosphorylated by CaMKII (Thr17; b ) or PKA (Ser16; c ) was downregulated in AF animals. d Phosphorylation levels relative to total PLN. e Serca2a protein analysis revealed a trend towards reduced protein expression associated with AF. Mean +- SEM optical density data normalized to GAPDH obtained from n = 5 Western blots per group are displayed. * P < 0.05; ** P < 0.01.

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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.

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Experimental details: Fig. 4 SGO1 interaction with HCN4. a SGO1-WT and SGO1-K23E transduction did not change HCN1, HCN2, HCN3, or HCN4 mRNA expression in NRVMs. n = 7 biologically independent preparations, each exposed in parallel to each condition. Data are expressed as mean +- SEM, normalized to respective control sample result for each preparation. b, c Transduction of SGO1-WT or SGO1-K23E did not affect HCN2 protein expression. n = 5 biologically independent preparations, each exposed in parallel to each condition. Data are expressed as mean +- SEM, normalized to respective control sample result for each preparation. d, e Transduction of SGO1-WT or SGO1-K23E did not change HCN4 protein expression. n = 5 biologically independent preparations, each exposed in parallel to each condition. Data are expressed as mean +- SEM, normalized to respective control sample result for each preparation. f Co-IP of native SGO1 with HCN4 in non-transduced NRVMs. NRVM lysates were incubated with HCN4 antibody or rabbit IgG (isotype antibody control); SGO1 antibody was used to detect SGO1 bands. Similar results were obtained in n = 5 biologically independent experiments. g A reverse co-IP with phospholamban (PLN) antibody as the isotypic antibody control. NRVM lysates were loaded as the input control in f and g . IP: immunoprecipitation. WB: Western blot. Similar results were obtained in n = 5 biologically independent experiments. h Confocal images from NRVM slides showing colocalization between native HCN4 and SG

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Experimental details: Figure 5 Effects of hyperaldosteronism and/or hypoestrogenism on calcium-handling regulatory proteins in wild type ( WT ) or aldostereone synthase overexpressing ( AS ) mouse hearts . (A) shows representative immunoblots of ryanodine receptor (RyR2), sarcoplasmic reticulum Ca 2+ - ATP ase ( SERCA 2a), phospholamban ( PLN ) and its phosphorylation (Thr17 or Ser16), and Na + /Ca 2+ -exchanger ( NCX ). (B-G) illustrate their respective quantification. The values expressed as mean +- SEM and normalized to GAPDH expression protein level ( n = 5-10 in each group).* P < 0.05; ** P < 0.01, and ### P < 0.001.

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Experimental details: Figure 4 Effect of pressure overload on RV cardiomyocytes and calcium handling. ( A ) cardiomyocytes passive tension; ( B ) cardiomyocytes active tension; ( C ) myofilaments calcium sensitivity; ( D ) Hill -coefficient; ( E ) the rate of tension redevelopment (Ktr); Protein quantification of: ( F ) sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase 2a (SERCA2a); ( G ) ratio of phospholamban phosphorylated (p-PLB) to total (PLB); ( H ) ratio of SERCA2a to phospholamban; ( I ) sodium-calcium exchanger (NCX); ( I ) Junctophilin; ( L ) representative western blot lanes (for representative purposes the image was cropped and edited) representative membrane protein loading. n = 5 for each group. Values are mean +- SEM, One-way ANOVA. Ba/Deb vs Sh: alpha p < 0.05; alphaalpha p < 0.01; alphaalphaalpha p < 0.001; Deb vs Ba: chi p < 0.05; chichichi p < 0.001.

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Experimental details: Fig. 6 Western blot of EC-coupling proteins in human-induced pluripotent stem cell cardiomyocytes (iPSC-CM). ( a ) Representative original Western blots after treatment with empagliflozin (EMPA) or vehicle control (control) for 2 or 8 weeks from 4 differentiation experiments (Diff.) from 2 healthy donors. GAPDH was used as loading control. ( b ) Mean protein expression levels (normalized to the respective control group at 2 or 8 weeks) in iPSC-CM ( n = 4 differentiations, matched groups are displayed with matched individual symbols) and effects of 2 and 8 weeks treatment with empagliflozin (EMPA) on ryanodine-receptor type 2 (RyR2), ( c ) sodium-calcium exchanger (NCX), ( d ) sarcoplasmic reticulum Ca 2+ ATPase (SERCA), ( e ) phosphorylated Ca 2+ -/calmodulin-dependent protein kinase II (pCaMKII), ( f ) Ca 2+ -/calmodulin-dependent protein kinase II (CaMKII), and ( g ) phospholamban (PLB). Groups were statistically analyzed using two-way ANOVA with Sidak's test for multiple comparisons

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Experimental details: Figure 4 Cardiac myocyte Ca 2+ -handling proteins in mouse mammary tumor virus-polyomavirus middle T antigen (MMTV-PyMT)- and MMTV-PyMT+ mice--phospholamban ( A ); SERCA2a (sarcoplasmic or endoplasmic reticulum calcium 2 ATPase) ( B ); phosphorilated phospholamban (p-phospholamban) ( C ); sodium/calcium exchanger (NCX) ( D ); and SERCA2a/phospholambam ratio ( E ). Western blot bands are shown for each protein. In the MMTV-PyMT+ mice, phospholamban protein expression was increased ( P =0.011) and SERCA2a protein expression was not different between the MMTV-PyMT+ and MMTV-PyMT- groups ( P =0.165). SERCA2a/phospholamban ratio was reduced in the MMTV-PyMT+ mice ( P =0.007). p-Phospholamban and NCX protein expression were decreased ( P =0.038 and P =0.017, respectively) compared with MMTV-PyMT- mice. Mann-Whitney test. Values are expressed as medians and interquartile ranges (IQRs). Animals per group: 7 MMTV-PyMT- and 7 MMTV-PyMT+.

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Experimental details: Fig. 2 Repeated subcutaneous injection of PLN ASO in mice with PLN R14 Delta/Delta prevents cardiac dysfunction and improves survival. a Experimental design of the PLN R14 Delta/Delta intervention study, consisting of ASO#27 treatment with a 4-week loading phase with weekly dosing of 100 mg/kg and a 20-week maintenance phase with dosing of 50 mg/kg once every 4 weeks. Data points for functional and biochemical assessments were obtained at treatment week 4 (T4) which was the primary endpoint, 15 (T15), 19 (T19), and 23 (T23) at the end of the study. b SYBR green qPCR results of cardiac Pln mRNA expression ( n = 3 for wild-type [WT] and n = 4 for PBS and PLN-ASO). c Semi-quantified western blot analysis of PLN protein expression using LV protein lysates. Intensities were normalized to total protein loading control and the PBS treated mice ( n = 3 per group). d Immunofluorescence of WT, PBS treated and PLN-ASO treated PLN R14 Delta/Delta mice after 4 weeks treatment. Sections were co-stained for cardiac Troponin T (green), PLN (red), and DAPI (blue). Note the lower presence of visible PLN aggregations (white arrows) and the higher structural integrity of the PLN-ASO treated hearts vs. PBS control. Stainings were performed in two animals per group. e Representative MRI images after 4 weeks of treatment. f Quantification of LVEF (left ventricular ejection fraction), LVESV (left ventricular end-systolic volume), and LVEDV (left ventricular end-diastolic volume) ( n = 12 for PBS T4,

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Experimental details: Fig. 3 Repeated subcutaneous injection of PLN ASO in Cspr3/Mlp -/- mice reverses signs and symptoms of heart failure. a Experimental design of the PLN-ASO Cspr3/Mlp -/- intervention study. Repeated studies were run with either PLN-ASO #27 (100 mg/kg on Days 0, 7, 14, and 21) or PLN-ASO #26_C (100 mg/kg on day 0, 50 mg/kg on days 7 and 14) (Supplementary Data 1 ). Echocardiography was performed at baseline (i.e., before treatment initiation), and at end of study (i.e., after 28 days of treatment), followed by termination of animals. Hemodynamic assessment was done between day 24 and 28. b Representative Western blot of LV protein lysates stained for PLN protein to detect the monomer and pentamer. Protein loading was normalized to GAPDH and total PLN protein were semi-quantified by calculating intensities relative to PBS treated control (PBS n = 10, PLN-ASO #26_C n = 10). c Individual echocardiography assessment and quantification of left ventricular ejection fraction (LVEF), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV) 28 days after treatment relative to baseline measurements. Cspr3/Mlp -/- mice with LVEF

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Experimental details: Figure 2 Histological and molecular analysis of PLN-R14del mice hearts. ( A ) Principal component analysis plot of RNA-Seq analysis of left ventricles of WT, PLN-R14 Delta/+ and PLN-R14 Delta/Delta mice of 3 or 8 weeks of age (n = 4 per group). 95% confidence ellipses of clusters are marked in grey. Information on genes contributing to variance in principle component 1 (PC1, x-axis) and principle component 2 (PC2, y-axis) is shown in Supplementary Fig. 1B . ( B ) qPCR measurements of left ventricular mRNA levels of Nppa (ANP), Nppb (BNP), and the ratio of Myh7 (beta-MHC) to Myh6 (alpha-MHC) of 8-week-old WT, PLN-R14 Delta/+ and PLN-R14 Delta/Delta mice (n = 4, 5, and 5, respectively). ( C ) Representative images of left ventricular sections stained with Masson's trichrome (scale bar = 70 mum) with ( D ) quantification of myocardial fibrosis in WT, PLN-R14 Delta/+ and PLN-R14 Delta/Delta mice hearts (n = 4, 5, and 5, respectively). ( E,F ) qPCR measurements of left ventricular mRNA levels of cardiac remodelling genes Col1a1 , Col1a2 , Col3a1 , Lgals3 (Gal-3), Timp1 , and Mmp2 of 8-week-old WT, PLN-R14 Delta/+ and PLN-R14 Delta/Delta mice (n = 4, 5, and 5, respectively). ( G ) Representative images of left ventricular sections of 8-week-old WT, PLN-R14 Delta/+ and PLN-R14 Delta/Delta mice stained with an anti-PLN antibody (red), wheat-germ agglutinin (WGA) (green), and DAPI (blue) (scale bar = 35 mum). ( H ) Western blot analysis of monomeric PLN proteins in RIPA-soluble and RI

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Experimental details: Figure 3 Cardiac functional, histological and molecular analysis of 12- to 20-month-old PLN-R14 Delta/+ mice. Echocardiographic analysis of left ventricular end-diastolic diameter ( A ), end-systolic diameter ( B ), fractional shortening ( C ), and global longitudinal strain ( D ) of 12-, 18- and 20-month-old WT and PLN-R14 Delta/+ mice (n = 9, 10, 6, 10, 5 and 9, respectively). ( E ) qPCR measurements of left ventricular mRNA levels of Nppa (ANP), Nppb (BNP), and the ratio of Myh7 (beta-MHC) to Myh6 (alpha-MHC) of 20-month-old WT and PLN-R14 Delta/+ mice (n = 6 and 10, respectively). ( F ) Representative images of left ventricular sections of 20-month-old WT and PLN-R14 Delta/+ mice stained with an anti-PLN antibody (red), wheat-germ agglutinin (WGA) (green), and DAPI (blue) (scale bar = 35 mum) or ( G ) stained with Masson's trichrome (scale bar = 70 mum) with ( H ) quantification of myocardial fibrosis (n = 6 and 9, respectively). ( I,J ) qPCR measurements of left ventricular mRNA levels of cardiac remodelling genes Col1a1 , Col1a2 , Col3a1 , Lgals3 (Gal-3), Timp1 , and Mmp2 of 20-month-old WT and PLN-R14 Delta/+ mice (n = 6 and 10, respectively). Gene expression values are corrected for Rplp0 (36B4) gene expression, and shown as fold change compared to age-matched WT. Myocardial fibrosis is presented as fold change compared to age-matched WT. Data are presented as mean +- S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001 compared to age-matched WT (Mann-Whitney test).

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Experimental details: Figure 2 IL-6 neutralization prevents atrial inflammation and the alterations in Ca 2+ handling proteins in SP rats. (A, B) Representative images of atrial MPO staining (A) and quantification (B) in the 4 indicated groups. Five visual fields are taken in each sample, and the number of immune cells from these fields is quantified with ImagePro 6.0 software and is averaged to make a statistical analysis. n = 6-7/group. Scale bar: 50 um. (C, D) Representative images of atrial CD68 staining (C) and quantification (D) . n = 6-7/group. Scale bar: 50 um. (E-I) Original Western blot (E) and quantification of the expression of RyR2, p-RyR2 (Ser2808), p-RyR2 (Ser2814) (F) , the expression of SERCA and NCX (G) , SERCA activity (H) , the expression of PLB, p-PLB (Ser16), and p-PLB (Thr17) (I) , in atrial tissue of 4 indicated groups. n =6-8/group. * P < 0.05, *** P < 0.001 vs . Sham; # P < 0.05, ## P < 0.01, ### P < 0.001 vs . IgG, determined by Student t-test or one-way ANOVA with Bonferroni's post-hoc test. (F-H) .

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Experimental details: Figure 8 Exogenous IL-6 administration increases the alterations in Ca 2+ handling proteins and AF induction in vivo . (A-E) Original Western blot (A) and quantification the expression of RyR2, p-RyR2 (Ser2808), p-RyR2 (Ser2814) (B) , the expression of SERCA and NCX (C) , SERCA activity (D) , the expression of PLB, p-PLB (Ser16), and p-PLB (Thr17) (E) , in atrial tissue of 2 indicated groups. n = 6/group. (F, G) Statistical results of AF duration (F) and probability (G) in the 2 indicated groups. n = 8/group. * P < 0.05, ** P < 0.01, *** P < 0.001 vs . Control determined by Student t-test.

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Experimental details: Fig. 4. Phospholamban is down-regulated at day 3. Sarcoplasmic reticulum Ca 2&plus pump (SERCA; A ) and phospholamban (PLB; B ) expression in mice at baseline, 12 h after lipopolysaccharide (LPS) administration, and at day 3. Upper panels show illustrative Western blots of SERCA, phospholamban, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in left ventricle extracts. Lower panels shown average data, normalized to GAPDH (loading control), and shown as a ratio of baseline. N = 6 hearts each. Phospholamban was visualized in the pentameric (25 kilodalton &lsqbkd&rsqb) and tetrameric forms (20 kd). Average data shown are for the predominant 20-kd isoform, and similar results were obtained for the 25-kd isoform. Data are shown as mean +- SD. BL = baseline. &ast P < 0.05 versus baseline.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2. PP1s differentially regulate substrate phosphorylation in neonatal cardiac myocytes Western blot analysis for the phosphorylation of PLB at serine 16 and threonine 17, total PLB, and PP1s from neonatal rat cardiac myocytes infected with adenoviruses expressing either beta-gal (Con) or individual PP1 isoform. p-MLC2 was performed using ProQ-Diamond staining method. Quantification was based on individual western blot. N = 4 for each group. * P < 0.05 vs. Con 5 min.