MA3-912

antibody from Invitrogen Antibodies

Targeting: ATP2A1 ATP2A, SERCA1

Provider product page for MA3-912

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [77]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [3]
Immunohistochemistry [2]
Other assay [37]

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Product number: MA3-912 - Provider product page
Provider: Invitrogen Antibodies
Product name: SERCA1 ATPase Monoclonal Antibody (VE121G9)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MA3-912 detects sarcoplasmic or endoplasmic reticulum calcium 1 (SERCA1) ATPase in canine, amphibian, human, mouse, rat, and rabbit tissues.
Antibody clone number: VE121G9
Concentration: 1 mg/mL

ATP2A1 protein structure - MA3-912 shown in red.

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Experimental details: Western blot analysis of SERCA1 ATPase was performed by loading 25 µg of human skeletal muscle (lane 1), rat skeletal muscle (lane 2) and mouse skeletal muscle (lane 3) onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4ºC overnight. The membrane was probed with a SERCA1 ATPase monoclonal antibody (Product # MA3-912) at a dilution of 1:2000 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 ~110 kDa.

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Experimental details: Western blot was performed using Anti-SERCA1 ATPase Monoclonal Antibody (VE121G9) (Product # MA3-912) and a 95 kDa band corresponding to SERCA1 ATPase was observed across Mouse and Rat skeletal muscle. Tissue extracts (30 µg lysate) of Mouse Skeletal Muscle (Lane 1), Rat Skeletal Muscle (Lane 2), Mouse Heart (Lane 3) and Rat Heart (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). 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 (1:2500) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

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Experimental details: Immunofluorescent analysis of SERCA1 ATPase using Anti-SERCA1 ATPase Monoclonal Antibody (VE121G9) (Product # MA3-912) shows staining in A2058 Cells. SERCA1 ATPase 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 SERCA1 ATPase (Product # MA3-912) at a dilution of 1:20 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.

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Experimental details: Immunofluorescent analysis of SERCA1 ATPase using Anti-SERCA1 ATPase Monoclonal Antibody (VE121G9) (Product # MA3-912) shows staining in Hela Cells. SERCA1 ATPase 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 SERCA1 ATPase (Product # MA3-912) at a dilution of 1:20 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.

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Experimental details: Immunofluorescent analysis of SERCA1 ATPase using Anti-SERCA1 ATPase Monoclonal Antibody (VE121G9) (Product # MA3-912) shows staining in U251 Cells. SERCA1 ATPase 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 SERCA1 ATPase (Product # MA3-912) at a dilution of 1:20 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.

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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 SERCA1 ATPase (Product # MA3-912) 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: 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 1:100 with a mouse monoclonal antibody recognizing SERCA1 ATPase (Product # MA3-912) 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 1. Capn3 deficiency in mouse C2C12 myotubes reduces SERCA protein levels and SERCAfunction. (a) C2C12 myotubes treated with NS or Capn3 shRNAs and differentiated for7 days. Scale bar = 100 mum (b) Western blot analysis showing significant decreaseof Capn3 (SPA antibody), SERCA1, SERCA2 and RyR1 levels in Capn3 knockdown myotubes,compared with controls (* P < 0.05;** P < 0.01). Total levels of DHPR, TRPC1, MyHC and actin arenot significantly changed. N = 3 independent experiments run on thesame gel. (c) SERCA-specific ATPase activity determined in homogenates from C2C12myotubes. Capn3-deficient myotubes show a significant reduction of SERCA activitycompared with NS-controls ( n = 3,* P < 0.05). (d and e) Ca 2+ imaging of C2C12myotubes loaded with Fura2-AM shows delayed Ca 2+ clearance from thecytosol in Capn3 knockdown myotubes. (d) Two representative traces of changes inFura2-AM fluorescence ratios( F 340 / F 380 ) fromCapn3-shRNA and NS-shRNA treated myotubes. Ca 2+ transients were elicitedby local stimulation with KCl 50 m m in the absence of extracellularCa 2+ . (e) Tau, the time constant of the Ca 2+ transient decayphase in seconds (s), is significantly increased in Capn3-deficient myotubes.** P < 0.01, n = total number of myotubesrecorded from six different experiments are shown in the graph.

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Experimental details: Figure 2. CAPN3 deficiency in human myotubes reduces SERCA protein levels and SERCA function.(a) LHCN-M2 myoblasts treated with control NS-shRNA or CAPN3-shRNAs anddifferentiated for 9 days. Scale bar = 50 mum. (b) Representative Western blotanalysis showing decrease of CAPN3 (12A2 mAb), SERCA1, SERCA2 and RyR1 proteinlevels in CAPN3-sh treated myotubes compared with controls. DHPRalpha2, MyHC and actinlevels remain unaltered. (c) SERCA-specific ATPase activity determined inhomogenates from LHCN-M2 myotubes. CAPN3-deficient myotubes show a significantreduction of SERCA activity compared with NS-controls ( n = 3,* P < 0.05). D-F) Ca 2+ imaging of LHCN-M2myotubes loaded with Fura2-AM shows increased cytosolic [Ca 2+ ] anddelayed Ca 2+ clearance from the cytosol in CAPN3 knockdown myotubes. (d)Resting cytosolic [Ca 2+ ] was measured in the presence of 2 m m Ca 2+ at 37degC. CAPN3-deficient human myotubes show significantlyincreased resting cytosolic [Ca 2+ ] compared with controls(* P < 0.05; n = 10 experiments). Totalnumbers of myotubes recorded are shown in the graph. (e) Two representative tracesof changes in Fura2-AM fluorescence ratios( F 340 / F 380 ) fromCAPN3-shRNA and NS-shRNA treated myotubes. Ca 2+ transients were elicitedby local stimulation with KCl 130 m m in the absence of extracellularCa 2+ . (f) Tau, the time constant of the Ca 2+ transient decayphase in seconds (s), is significantly increased in CAPN3-deficient myotubes.* P < 0.05, n = total number of myotubesr

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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.

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Experimental details: Figure 1. Capn3 deficiency in mouse C2C12 myotubes reduces SERCA protein levels and SERCAfunction. (a) C2C12 myotubes treated with NS or Capn3 shRNAs and differentiated for7 days. Scale bar = 100 mum (b) Western blot analysis showing significant decreaseof Capn3 (SPA antibody), SERCA1, SERCA2 and RyR1 levels in Capn3 knockdown myotubes,compared with controls (* P < 0.05;** P < 0.01). Total levels of DHPR, TRPC1, MyHC and actin arenot significantly changed. N = 3 independent experiments run on thesame gel. (c) SERCA-specific ATPase activity determined in homogenates from C2C12myotubes. Capn3-deficient myotubes show a significant reduction of SERCA activitycompared with NS-controls ( n = 3,* P < 0.05). (d and e) Ca 2+ imaging of C2C12myotubes loaded with Fura2-AM shows delayed Ca 2+ clearance from thecytosol in Capn3 knockdown myotubes. (d) Two representative traces of changes inFura2-AM fluorescence ratios( F 340 / F 380 ) fromCapn3-shRNA and NS-shRNA treated myotubes. Ca 2+ transients were elicitedby local stimulation with KCl 50 m m in the absence of extracellularCa 2+ . (e) Tau, the time constant of the Ca 2+ transient decayphase in seconds (s), is significantly increased in Capn3-deficient myotubes.** P < 0.01, n = total number of myotubesrecorded from six different experiments are shown in the graph.

Supportive validation

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Experimental details: Figure 2. CAPN3 deficiency in human myotubes reduces SERCA protein levels and SERCA function.(a) LHCN-M2 myoblasts treated with control NS-shRNA or CAPN3-shRNAs anddifferentiated for 9 days. Scale bar = 50 mum. (b) Representative Western blotanalysis showing decrease of CAPN3 (12A2 mAb), SERCA1, SERCA2 and RyR1 proteinlevels in CAPN3-sh treated myotubes compared with controls. DHPRalpha2, MyHC and actinlevels remain unaltered. (c) SERCA-specific ATPase activity determined inhomogenates from LHCN-M2 myotubes. CAPN3-deficient myotubes show a significantreduction of SERCA activity compared with NS-controls ( n = 3,* P < 0.05). D-F) Ca 2+ imaging of LHCN-M2myotubes loaded with Fura2-AM shows increased cytosolic [Ca 2+ ] anddelayed Ca 2+ clearance from the cytosol in CAPN3 knockdown myotubes. (d)Resting cytosolic [Ca 2+ ] was measured in the presence of 2 m m Ca 2+ at 37degC. CAPN3-deficient human myotubes show significantlyincreased resting cytosolic [Ca 2+ ] compared with controls(* P < 0.05; n = 10 experiments). Totalnumbers of myotubes recorded are shown in the graph. (e) Two representative tracesof changes in Fura2-AM fluorescence ratios( F 340 / F 380 ) fromCAPN3-shRNA and NS-shRNA treated myotubes. Ca 2+ transients were elicitedby local stimulation with KCl 130 m m in the absence of extracellularCa 2+ . (f) Tau, the time constant of the Ca 2+ transient decayphase in seconds (s), is significantly increased in CAPN3-deficient myotubes.* P < 0.05, n = total number of myotubesr

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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

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Experimental details: Figure 3 Representative western blots of the proteins investigated in slow-twitch (ST), fast-twitch (FT), and muscle homogenate (HOM) before (PRE) and after (POST) 7 weeks of high intensity and reduced volume training in trained cyclists. See methods section for details.

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Experimental details: Figure 5. Interaction of CAPN3 with SERCA1, SERCA2 and sAnk1 in human skeletal muscle. (a)Immunoprecipitation (IP) of CAPN3 with a goat polyclonal antibody (pIS2C) in a vastus lateralis muscle from a healthy donor. Both, SERCA1 andSERCA2 are detected in the CAPN3 IP. White lines indicate noncontiguous lanes run onthe same gel. Input: protein extract. (b) SERCA1 and SERCA2 were immunoprecipitated(IP) with specific monoclonal antibodies in the same muscle. sAnk1 is detected inSERCA1 IP but not in SERCA2 IP. (c) Longitudinal sections from a dorsal human muscleco-immunostained for CAPN3-SERCA1, CAPN3-SERCA2 and CAPN3-sAnk1, showing similardistribution pattern of CAPN3 (green), SERCA1 (red), SERCA2 (red) and sAnk1 (red) inthe panels labelled ""Merge"". Scale bar: 10 um. (d) Co-localisation analysis ofCAPN3, with SERCA1, SERCA2 and sAnk1 in longitudinal sections from human dorsalmuscle using in situ PLA. Red spots represent protein complexes in close proximity(

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Experimental details: Figure 6. Increased protein degradation of SERCA1 and SERCA2 in CAPN3-deficient LHCN-M2 humanmyotubes. (a) CAPN3 and SERCA mRNA levels were analysed in human myotubes byquantitative RT-PCR. Statistical analysis showed significant decrease in CAPN3expression (19.3 +- 2.6%) in CAPN3 knockdown myotubes as compared to matched controls(100 +- 12.8%). N = 3; * P < 0.005. Nosignificant changes were observed in SERCA1, SERCA2 or SERCA3 mRNA expressionlevels. (b) SERCA1 and (c) SERCA2 ubiquitination and sumoylation were analysed incontrol and CAPN3-deficient human myotubes through SERCA1/2 immunoprecipitation withspecific mouse monoclonal antibodies. Pools of control and CAPN3-deficient cultureswere used for immunoprecipitation assays. N = 2 and N = 3 independent experiments were performed for SERCA1 and SERCA2,respectively. White lines indicate noncontiguous lanes run on the same gel.Ubiquitination of SERCA1 and SERCA2 in CAPN3-deficient myotubes was increased 3.30and 4.35-fold, respectively, compared with controls. No SUMO1 specific sumoylationof SERCA1 and SERCA2 proteins was detected in controls or CAPN3-deficientmyotubes.

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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;

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Experimental details: Figure 5 Decreased activity of Serca1 and increased oxidative stress in naturally and prematurely aged gastrocnemius muscle. (a) Significant reductions occurred in calcium-dependent Serca ATPase activity in naturally aged (26M) WT and prematurely aged (3M) Cisd2 mKO mice. The selective Serca pump inhibitor, TBQ, was used to reflect a specific difference in Serca activity. n = 4 for each group of mice. (b) Increased cysteine S-sulfonation and tyrosine nitration on Serca1 protein in gastrocnemius muscles. The total Serca1 protein was detected by re-blotting on the same membrane. (c,d) Increase in oxidative modifications, namely S-sulfonated cysteine and 3-nitrotyrosine, for all proteins present in whole cell extracts of the gastrocnemius muscle. Quantification of each sample was based on the entire intensities of each lane or region normalized to beta-tubulin. *p < .05

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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.

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Experimental details: 2 FIGURE Sarco(endo)plasmic reticulum Ca 2+ ATPase (SERCA) function is impaired and SERCA2a T-nitration is increased in soleus muscles of SOD2 +/- mice. (a) SERCA activity- p Ca curves in wild-type (WT) and SOD2 +/- mice over Ca 2+ concentrations ranging from p Ca 6.83-6.10, presented as % of V max . p Ca 50 values are embedded in the graph with 95% confidence intervals (CIs). (b) No differences in maximal SERCA activity ( mu mol/g of protein/min) were observed between genotypes. (c) Densitometric analysis and representative images of Western blots for SERCA2a, SERCA1a, and sarcolipin protein content as well as analyses and representative images of SERCA-specific T-nitration (d). All values are mean +- SEM and are presented relative to WT. * p < 0.05 ** p < 0.01, values above bars indicate p values using Student's t -test ( n = 5-8 per group).

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Experimental details: 10.1371/journal.pone.0264146.g001 Fig 1 Effect of triadin ablation on the expression of couplon proteins. A , Western blot of proteins of interest from WT and Tr- total homogenates. Band signal mass was normalized to signal in the corresponding lane of the gel in B (boxed region a corrected for baseline level in b ). Details in Methods and []. B .1, Ponceau-stained proteins separated by PAGE. B .2, uncropped blot showing immunoreactivity of triadin antibody. C , distribution of results in Tr- samples as a ratio of the WT average. Different symbols identify values obtained from individual mice. Error bars represent SEM. Asterisks mark changes statistically significant at the 0.05 (*) level. Statistical parameters are listed in Table 1 .

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Experimental details: FIGURE 2 Effect of UPS inhibition in myotubes from LGMDR1 patients. (A) Representative bright-field images showing immortalized human myotubes from a control and two LGMDR1 patients (LG1 and LG2) after 5 days in differentiation. Scale bar: 50 mum. (B) Cumulative distribution of myotube width from control, LG1 and LG2 myotubes. 90-170 myotubes analyzed from n = 3 independent experiments. Data are expressed as mean % +- SEM (two-way ANOVA test). (C) Western blot analysis of CAPN3, SERCA1, SERCA2, RyR1, and DHPRa2. Quantification of protein signals is shown below each blot, represented as fold change over control. (D) qPCR expression analysis of Ca 2+ -handling proteins in myotubes from control and LGMDR1 patients, treated or not with BTZ. Data are expressed as mean +- SEM. n = 3 independent experiments. * p < 0.05, One-way ANOVA post hoc Tukey's multiple comparisons test. (E) Representative western blot signals from LG1 and LG2 myotubes treated with 5 nM BTZ for 24 h after 5 days of differentiation and their quantification. Protein signals are normalized to total protein (gel load) and represented by fold change over non-treated LGMDR1 myotubes. Non-treated LGMDR1 SERCA expression levels are shown as a discontinuous line.

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Experimental details: Qualitative and quantitative EM of SR and WB analysis of SERCA-1 expression. ( A - C ) Representative EM images of EDL muscle fibers from WT (panel ( A )), CASQ1- (panel ( B )), and Y522S (panel ( C )) mice showing longitudinal SR cisternae appearance (empty arrows). ( D ) Quantitative analysis of the relative fiber volume occupied by the SR. ( E , F ) Representative immunoblots (panel ( E )) and relative band densities normalized to GAPDH levels of SERCA-1 expression (panel ( F )) in EDL muscle homogenates. Data are given as mean +- SEM (* p < 0.05), as evaluated by chi-squared test with Yates's correction for continuity (panel ( D )) or by one-way ANOVA followed by Tukey's post hoc test (panel ( F )). Scale bar ( A - C ), 1 um. n = number of mice.

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Experimental details: Phospholamban is reduced in the soleus muscles of Ng +/- mice. Representative Western blots and their corresponding analyses for SERCA2a, SERCA1a, RYR, SLN and NNAT in the soleus of WT and Ng +/- mice (A) . Only reductions in SERCA1a content were observed, though this did not reach statistical significance. Characterizing PLN and its activation status showed reductions in monomeric PLN resulting in reductions in the monomer:pentamer ratio and statistically insignificant increases in the p-PLN : PLN ratio (B) . Molecular weight markers are included in the representative blots. * p < 0.05, values above bars indicate p values (n=4-5 per group).

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Experimental details: Figure 5 SERCA content is reduced in iWAT and BAT from Ng +/- mice. Representative Western blots and corresponding analyses of SERCA2a, SERCA1a, and UCP1 from iWAT (A) and BAT (B) . In both depots, reductions in both SERCA isoforms were observed with no changes in UCP1. * p < 0.05, values above bars indicate p values (n=9-10 per group).