Stim1

All protein samples were extracted from mice hearts. Protein concentrations were determined using a Bradford assay with bovine serum albumin (BSA) as a standard. To determine the protein expression of STIM1 (BD Biosciences, CA, USA), STIM2 (Cell Signaling Technology, MA, USA), TRPC1, TRPC3, TRPC4, and TRPC6 (Alomone Labs, Jerusalem, Israel), pNFATc4 (rabbit anti-mouse polyclonal antibody raised against short amino acid sequence containing Ser 168 and 170 dually phosphorylated NFATc4 of human origin, Santa Cruz Biotechnology, TX, USA), NFATc4 (rabbit anti-mouse polyclonal antibody raised against amino acids 125–198 of NFATc4 human origin, Santa Cruz Biotechnology, TX, USA), samples (50 μg) were run on 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes, and rocked at 4°C for 1 h in blocking buffer (0.1% Tween 20 and 1% BSA in Tris-buffered saline). An enhanced chemiluminescence (ECL)-detection system was used to detect the bound antibodies. An anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Santa Cruz Biotechnology, TX, USA) was used as an internal loading control.

All RVOT myocytes were centrifuged, washed with cold phosphate‐buffered saline and lysed on ice for 30 min in an RIPA buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktails (Sigma‐Aldrich Corp.). The protein concentration was determined using a Bio‐Rad protein assay reagent (Bio‐Rad). Proteins were separated using 10% SDS‐PAGE under reducing conditions and electrophoretically transferred into an equilibrated polyvinylidene difluoride membrane (Amersham Biosciences). All blots were probed using primary antibodies against SERCA2a, NCX (Swant), CaMKII, phosphorylated CaMKII at Thr 286 (pCaMKII), ryanodine receptor (RyR) channels, PKA, connexin 43, stromal interaction molecule 1 (STIM1; BD BioScience), phosphorylated phospholamban at Ser16 (pPLB S16; GeneTex), mature collagen α1 type I (Santa Cruz Biotechnology), phosphorylated RyR at Ser2808 and Ser2814 (pRyR S2808 and pRyR S2814), phosphorylated PLB at Thr 17 (pPLB T17; Badrilla), PLB (Thermo), GADPH (MBL) and all secondary antibodies conjugated with horseradish peroxidase. All bound antibodies were detected using an enhanced chemiluminescence detection system and analysed using AlphaEaseFC software. All targeted bands were normalized to GADPH to verify the presence of equal protein loading.

Cell lysates were harvested in RIPA buffer (150 mM NaCl, 1 mM EGTA, 50 mM Tris at pH 7.4, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and Complete^TM), and subsequently analyzed by Western blotting using antibodies against STIM1, FAK, paxillin, E-cadherin, ZO-1, fibronectin (BD Biosciences, San Jose, CA), Orai1, Orai3 (Prosci, Poway, CA), Orai2, phospho-Tyr397-FAK (Enzo, Farmingdale, NY), TRPC1 (Proteintec, Rosemont, IL), vinculin, vimentin, N-cadherin (Santa Cruz, Santa Cruz, CA), talin (abcam, Cambridge, UK), phospho-Tyr18-paxillin (Invitrogen, San Diego, CA), STIM2 (Cell Signaling, Danvers, MA), and β-actin (Sigma-Aldrich, Saint Louis, MO). The immunocomplexes were then detected with horseradish peroxidase-conjugated IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), and the reaction was developed using an ECL detection kit (Amersham, Piscataway, NJ) under an ImageQuant LAS 4000 system (GE Healthcare Life Sciences, Pittsburgh, PA).