Anti mouse or anti rabbit secondary antibodies

HL-1 cardiomyocytes or human tissue samples were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Cell Signaling) supplemented with protease and phosphatase inhibitors (complete Mini and PhosSTOP; Roche) on ice for 5-15 min. After centrifugation to pellet nondissolved material, the protein concentration of each sample was determined using the bicinchoninic acid method (BCA; Thermo Fisher).
Equal amounts of protein homogenates were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Membranes were blocked in blocking buffer and probed with the primary antibody (overnight at 4°C). The following primary antibodies were used: RYR2 (2446725; Millipore), p21 (ab109199; ABCAm), p53 (2524; Cell Signaling), p16 (ab211542; ABCAm), γH2AX (9718; Cell Signaling), CACANA1C (Cav1.2; ab84814; ABCAm), SERCA2 (ab150435; ABCAm), and PLB (ab219626; ABCAm). The membranes were subsequently incubated with anti-mouse or anti-rabbit secondary antibodies (ABCAm). Signals were detected using the enhanced chemiluminescence (ECL) detection method (Thermo Fisher) and quantified by densitometry (ImageJ). The original uncropped blots are available in the Supplementary Information section.

Total protein samples were extracted from NSCLC cells and tumor tissues collected from human or nude mice using RIPA lysis buffer (Beyotime, Jiangsu, China, P0013K) and protease inhibitor cocktail (Roche, Germany, 04963159001) at a ratio of 100:1. Electrophoretic experiments were performed with an equal amount of protein, and the samples were transferred to the nitrocellulose blotting membrane (Merck Millipore, R7BA46025). The membrane was blocked with the Western Quick Block Kit (NCM Biotech, Suzhou, China) for 10 min and probed with primary antibodies against fibronectin 1 (FN1, Proteintech, Rosemont, IL, USA, 15613-1-AP 1:1000), ZO-1 (Proteintech, 21773-1-AP, 1:500), E-cadherin (Proteintech, 20874-1-AP, 1:500), YAP1 (Proteintech, 13584-1-AP, 1:1000), Vimentin (Cell Signaling, #5741, 1:1000), Gankyrin (Abcam, Cambridge, UK, ab182576, 1:1000) and β-actin (Proteintech, 66009-1-Ig, 1:10,000) overnight at 4 °C. The membranes were then incubated with anti-mouse or anti-rabbit secondary antibodies (Abcam, Cambridge, UK, 1:10,000) for 50 min at room temperature. Finally, immunoreactivity was measured using the Odyssey Infrared Imaging System (Odyssey, LICOR, USA).