Co-immunoprecipitation Assay of Mouse Myotubes

Mouse primary skeletal myotubes were solubilized in lysis buffer, as previously described^{6 (link),20 (link),27 (link),40 (link),63 (link),64 (link)}. For the co-immunoprecipitation assay^{20 (link),40 (link),63 (link)}, solubilized myotube lysate (80 or 100 µg of total protein) was used. The immunoprecipitate was subjected to immunoblot assays with anti-DHPR, anti-STIM1, or anti-GFP antibody (Abcam, Cambridge, MA, U.S.A., for detecting CFP-R429C). For the immunoblot assays, the solubilized myotube lysate (10 μg of total protein) was subjected to SDS-PAGE (8, 10, or 12% gel)^{6 (link),20 (link),27 (link),40 (link),63 (link),64 (link)}. Anti-RyR1, anti-SERCA1a, anti-CSQ1, anti-CaM1, anti-MG29, anti-MG53, anti-JP1, and anti-JP2 antibodies were obtained from Affinity BioReagents (Golden, CO, U.S.A.). Anti-TRPC1, anti-TRPC3, anti-TRPC4, and anti-TRPC6 antibodies were obtained from Alomone Laboratories (Jerusalem, Israel). Anti-Orai1, anti-STIM1, anti-STIM2, and anti-α-actin antibodies were obtained from Abcam. Anti-Drp-1, anti-Mfn1, anti-calcineurin and anti-CaMKII antibodies were obtained from Santa Cruz Biotechnology (Paso Robles, CA, U.S.A.).