Co-immunoprecipitation and Western blotting to detect the relative amount of FKBP12.6 associated with RyR2

We examined FKBP12.6 association with RyR2 as described previously (37, 38). Briefly, cardiomyocytes were lysed in modified radioimmune precipitation lysis buffer, shaking on ice for 20 min. The lysates were centrifuged at 12,000 × g for 15 min at 4 °C. The supernatants were collected, and the protein concentrations were determined with a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). The 100 μg of supernatant protein was incubated with 2 μg of anti-RyR2 antibody (Abcam) in 0.1 ml of modified radioimmune precipitation buffer and shaken slowly overnight at 4 °C. The samples were incubated with 40 μl of protein A/G-agarose beads at 4 °C for 3 h. The resins were washed three times with radioimmune precipitation buffer, and the eluted immunoprecipitated proteins were boiled for 5 min at 95 °C and loaded into wells in the 10% SDS-PAGE before being transferred to PVDF membranes and then probed with primary antibody: anti-RyR2 (1:1000; Abcam), FKBP12.6 (1:2000; Elabscience). Bound antibodies were visualized using the enhanced chemiluminescence (ECL) detection kit (Beyotime). The FKBP12.6 associated with RyR2 was calculated as the ratio of FKBP12.6 to RyR2 protein content in RyR2 immunoprecipitates. Total FKBP12.6 protein level in cell lysates was detected as input to indicate the expression level of FKBP12.6.