Anti dhpr

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.

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.).

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).