Anti β mhc antibody

Protein samples were extracted from tissues and cells with the same procedures described previously. Briefly, the heart tissues and myocardial cells were lysed in RIPA buffer and then centrifuged at 4°C 12 000 g for 15 minutes. The supernatant was collected, and protein concentrations were determined by bicinchoninic acid (BCA) protein assay (Beyotime, Shanghai, China). Protein extracts were separated by SDS‐PAGE and transferred onto a nitrocellulose membrane. Then, the membranes were blocked and incubated with the following primary antibodies: anti‐BNP antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐β‐MHC antibody (1:2000, Sigma, St Louis, MO, USA), anti‐TRPV3 antibody (1:200, Abcam, Cambridge, MA, USA), anti‐Beclin‐1 antibody (1:500, Abcam), anti‐LC3‐II antibody (1:500, Abcam)and anti‐β‐actin (1:2000, Santa Cruz Biotechnology). β‐actin was used as a loading control. After incubation at 4°C overnight, membranes were washed for three times with Tris Buffered Saline with Tween‐20 (TBST), 10 minutes each time, then incubated with secondary antibody for 1 hour at room temperature in the dark. Finally, the membranes were washed for three times with TBST, 10 minutes each time, and scanned by Odyssey Imaging System (LI‐COR Biosciences, Lincoln, NE, USA).