Anti fundc1

The LV tissue and cardiomyocytes were washed with cold PBS and lysed with RIPA buffer containing a protease inhibitor cocktail. Lysates were centrifuged at 14,000 × g for 15 min at 4°C. Protein concentration was quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). The proteins in the lysate (10–60 μg) were separated by electrophoresis, and the bands were transferred to PVDF membranes. The membranes were incubated for 2 h in 5% nonfat dry milk to block nonspecific antibody binding to the membrane and then overnight at 4°C with primary antibodies. The primary antibodies used were as follows: anti-Fundc1 (1 : 1,000, Abcam, #ab224722), anti-LC3B (1 : 1,000, Cell Signaling Technology, #2775S), anti-Pink1 (1 : 1,000, Abcam, #ab23707), and anti-Parkin (1 : 1,000, Proteintech, #23272-1-AP). The membranes were washed with Tris-buffered saline solution containing 0.1% Tween 20 (TBST, pH 7.6) and then probed with appropriate secondary antibodies (1 : 1,0000, Cell Signaling Technology, #5151S or #5257S) at room temperature for 90 min. For use as internal controls, GAPDH and β-actin levels were quantified using specific antibodies (anti-GAPDH, 1 : 1,0000, Proteintech, #10494-1-AP; anti-β-actin, 1 : 1,000, Cell Signaling Technology, #4970S). The protein bands were detected using a Bio-Rad imaging system (Hercules, CA, USA).