Bm chemiluminescence blotting system

Subsequently, using the RIPA buffer, proteins were extracted from cells for immunoblotting. 15–50 μg of total protein extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After blocked with 5% skimmed milk, membranes were then probed with anti-Bax (1:2,000 dilution; #ab3203; Abcam, Cambridge, UK), anti-Bcl-2 (1:1,000; #ab32124; Abcam), anti-cleaved-caspase3 (1:500; #ab32042; Abcam), anti-cleaved-caspase9 (1:1,000; #ab2324; Abcam), anti-α-SMA (1:1,000; #ab5694; Abcam), anti-SM22 (1:1,000; #ab14106; Abcam), anti-SM-MHC (1:2,000; #ab133567; Abcam), anti-vimentin (1:2,000; #ab92547; Abcam), anti-collagen I (1:1,000; #ab270993; Abcam), anti-SMAD3 (1:2,000; #ab40854; Abcam), and anti-β-actin (1:5,000; #ab8226; Abcam) at room temperature for 1.5 h. Then, membranes were incubated with the appropriate secondary antibody conjugated to HRP. Then, the BM chemiluminescence blotting system (Thermo Fisher Scientific, Waltham, MA, USA) was used to visualize protein bands, and ImageJ Software (NIH, Bethesda, MD, USA) was used to quantify protein bands.