M200006

Cells and tissues were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio) with a protease-inhibitor cocktail (Roche) and a phosphatase- inhibitor cocktail (Roche). Total protein concentrations were normalized by BCA without boiling. Western blot was performed using standard protocol with the following primary antibodies: RyR2 (Abcam, ab2868, 1:1000), RyR2-phospho Ser2814 (Badrilla, A010-31AP, 1:500), CaMKII (Abcam, ab181052, 1:1000), CaMKII-phospho T286 (Abcam, ab32678, 1:1000), Ca_v1.2 (Abcam, ab84814, 1:1000), NCX1 (Proteintech, 55075-1-AP, 1:1000), SERCA2a (Santa Cruz Biotechnology, sc-53010, 1:200), and GAPDH (Abmart, M200006, 1:5000).

The iPSC-CMs were detached with TrypLE and then, pelleted at 1000 rpm for 5 min at 4 °C. After washing with PBS, the pellets were re-suspended in 50–100 μl lysis buffer. Lysates were placed on ice for 30 min, and then, the supernatants were collected after centrifuging at 12,000 rpm for 15 min. Cytosolic and nuclear fractions were extracted with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific, 78,833) according to the manufacturer’s instruction. Protein concentration was measured using a BCA kit (Pierce, 23227). Western blot was performed using standard protocol with the following antibodies: Na_v1.5 (Alomone Labs, ASC-005, 1:500), total β-catenin (Abcam, ab32572, 1:5000), active β-catenin (Abcam, ab246504, 1:1000), Lamin B1 (Abcam, ab16048, 1:5000) and GAPDH (Abmart, M200006, 1:5000). Intensity values for each band were determined as the integrated density (sum of pixel values) within a fixed area using Quantity One software (Biorad).