Anti stim1

Mouse primary skeletal myotubes were solubilized in lysis buffer, as previously described [10 (link),22 (link),38 (link),41 (link),42 (link),44 (link)]. For the coimmunoprecipitation assay [22 (link),41 (link),42 (link),44 (link)], solubilized myotube lysate (100 µg of total protein) and anti-CASQ1 (Affinity BioReagents, Golden, CO, USA) or anit-Orai1 antibody (Abcam, Cambridge, MA, USA) were used. The immunoprecipitate was subjected to immunoblot assays with anti-CASQ1, anti-Orai1, or anti-STIM2 antibody (Abcam). For the immunoblot assay, solubilized myotube lysate (10 μg of total protein) was subjected to SDS–PAGE (8, 10, or 12% gel) [10 (link),22 (link),38 (link),41 (link),42 (link),44 (link),46 (link)]. The anti-RyR1, anti-SERCA1a, anti-CASQ1, anti-CaM1, anti-JP1, and anti-JP2 antibodies were obtained from Affinity BioReagents. The anti-TRPC1, anti-TRPC3, anti-TRPC4, and anti-TRPC6 antibodies were obtained from Alomone Laboratories (Jerusalem, Israel). The anti-TRIM32, anti-MyoD, and anti-myogenin antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The anti-DHPR, anti-STIM1, anti-STIM2, and anti-α-actin antibodies were obtained from Abcam.

For the co-immunoprecipitation assay, the solubilized triad sample (50 µg of total protein) was incubated with anti-STIM2 antibody (Sigma-Aldrich) overnight at 4 °C, as previously described^{26 (link),80 (link),81 (link),83 (link)}. Anti-STIM1 (Abcam, Cambridge, MA, USA), anti-SERCA1a (Thermo Scientific Inc., Rockford, IL, USA), or anti-TRPC6 (Alomone Laboratories, Jerusalem 9104201, Israel) antibody was used for immunoblot assay. For the immunoblot assay, fully differentiated mouse primary skeletal myotubes on 10-cm plates on differentiation day 5 were solubilized, and the solubilized lysate (5 or 10 μg of total protein) was subjected to SDS-PAGE (8, 10, or 12% gel) and immunoblot assay, as previously described^{15 (link),26 (link),45 (link),80 (link),83 (link),87 (link)}. Anti-RyR1, anti-CSQ, anti-CaM1, anti-JP1, and anti-JP2 antibodies were obtained from Thermo Scientific Inc. Anti-TRPC1, anti-TRPC3, and anti-TRPC4 antibodies were obtained from Alomone Laboratories. Anti-DHPR, anti-Orai1, and anti-α-actin antibodies were obtained from Abcam. Horseradish peroxidase-conjugated anti-goat, anti-mouse, or anti-rabbit secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The membranes were washed three times with PBS and developed using a SuperSignal Ultra Chemiluminescent substrate (Pierce, Rockford, IL, USA).

For immunocytochemistry assays, myotubes were fixed in cold methanol (−20 °C) for 30 min and permeabilized with 0.05% Tween 20 in PBS for 1 min, as previously described [7 (link),8 (link),16 (link),17 (link)]. For immunoblot assays, solubilized lysate of myotubes (15 μg of total protein) was subjected to SDS–PAGE (8 or 10% gel), as previously described [7 (link),8 (link),16 (link),17 (link),18 (link),19 (link),20 (link)]. Anti-RyR1 and anti-SERCA1a antibodies were obtained from Affinity BioReagents. Anti-DHPR, anti-Orai1, anti-STIM1, and anti-α-actin antibodies were obtained from Abcam (Cambridge, MA, USA).

All DNA constructs were generated by a PCR-based method and sequenced to confirm their fidelity. Orai1 and STIM1 were amplified from Orai1 (MMM1013-20276444), and STIM1 (MMM1013-202764946) cDNA purchased from Open Biosystems and introduced into pEBB vectors. For Orai1-CFP and STIM1-YFP vector construction, CFP and YFP were amplified from Raichu-Rac1 [25 (link)] and C-terminally introduced into pEBB-Orai1 and pEBB-STIM1, respectively. Anti-Flag (Sigma, F1804, St. Louis, MO, USA), anti-Orai1 (Santa Cruz, sc-68895, Dallas, TX, USA), anti-Orai1 (Abcam, ab111960, Cambridge, UK), anti-STIM1 (Abcam, ab108994), anti-IP₃R (Cell Signaling, #8568, Boston, MA, USA), anti-phospho-IP₃R (Cell Signaling, #3760S), anti-PLCγ1 (Cell Signaling, 2822S), anti-phospho-PLCγ1 (Cell Signaling, 2821S), anti-Mer (R&D systems, AF591, Minneapolis, MN, USA), and anti-β-Actin (Santa Cruz, sc-1616) were purchased.