Protein Expression Analysis by Western Blot

Western blot analysis was applied to determine the levels of protein expression in cells according to previous studies^{25 (link)–28 (link)}. Briefly, the proteins in the four groups were extracted by RIPA buffer (Beyotime, China) and transfected onto the PVDF membranes, then the PVDF membranes were incubated with 1:500 diluted mouse anti-p38 primary antibody (Abcam, UK), 1:500 diluted mouse anti-p-p38 primary antibody (Santa Cruz, USA), 1:500 diluted mouse anti-α-SM-actin primary antibody (Abcam, UK) and 1:1000 diluted rabbit anti-β-actin primary antibody (Sigma-Aldrich Co., USA) overnight at 4 °C. The following day, the membranes were probed with the 1:5000 IRDye diluted 700-conjugated affinity-purified goat anti-mouse second antibody (Rockland Immunochemicals, USA) or 1:5000 diluted IRDye 800-conjugated affinity-purified goat anti-rabbit second antibody (1:5000, Rockland Immunochemicals, USA) for 60 min at room temperature. Then protein bands were visualized using Odyssey laser scanning system (LI-COR Inc., USA) and the intensities were quantified by Odyssey 3.0 image analysis system software.