Western blotting to determine changes of protein or acetylation levels

At the end of each treatment, cells were collected and lysed in radio-immunoprecipitation assay buffer containing protease inhibitors (50 mmol/l Tris, 150 mmol/l NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, and 10 μg/ml aprotinin). Approximately 15 to 30 μg of proteins from each sample (quantified with a Bio-Rad DC Protein Assay kit, Bio-Rad, Hercules, California) were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 10% gel. The proteins were then transferred to a polyvinylidene difluoride membrane and detected by immunoblotting using respective antibodies. The antibody sources and dilution ratios are as follows: rabbit monoclonal anti-α-SMA, Abcam (Cambridge, Massachusetts; catalog no. ab32575), 1:1,000; rabbit polyclonal anti-SMHC, Abcam (catalog no. ab53219), 1:1,000; mouse monoclonal anti–β-actin, Abcam (catalog no. ab6276), 1:1,000; rabbit polyclonal anti–MRTF-A, Cell Signaling Technology (Danvers, Massachusetts; catalog no. 14760S), 1:1,000; rabbit polyclonal anti–Ac-Lysine, Cell Signaling Technology (catalog no. 9441S), 1:1,000; and rabbit polyclonal anti–Ac-αtubulin, Cell Signaling Technology (catalog no. 5335S), 1:1,000. Secondary antibodies were as follows: Goat Anti-Mouse IgG (H + L)-HRP Conjugate (Bio-Rad, Hercules, California, catalog no. 1706516) and Goat Anti-Rabbit IgG (H + L)-HRP Conjugate (Bio-Rad, catalog no. 1706515). Specific protein bands on the blots were illuminated by applying enhanced chemiluminescence reagents (Thermo Fisher Scientific; Catalog no. 32106) and then recorded with an Azur LAS-4000 Mini Imager (GE Healthcare Bio-Sciences, Piscataway, New Jersey). Band intensity was quantified by using ImageJ software.