Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Cells were lysed in RIPA buffer (Beyotime, China) with 1% PMSF (Biosharp, China). The protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Epizyme, China) and transferred onto PVDF membrane (Millipore, USA). Primary antibodies were applied at 4 °C overnight and HRP-conjugated secondary antibodies were applied for an hour at room temperature. The immunocomplexes were detected with ECL Western Blotting Substrate (NCM Biotech, China), visualized with Tanon (5200multi 4600SF, Tanon, USA). GAPDH was used as the internal control. Primary Antibodies included rabbit antiMAML3 (1:500, Biorbyt, UK), mouse antiGAPDH (1:20000, Beyotime, China). Secondary antibodies (A0208 and A0216, Beyotime, China) were diluted in 1:1000.

Proteins were removed from rat aortic SMCs following lysis and after intervention. The cells were divided into four groups, as described, and 40 µg of protein per lane was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Epizyme, Beijing, China), transferred to a polyvinylidene difluoride membrane (Millipore Sigma, Billerica, MA, USA), and blocked with western quick-blocking buffer (Beyotime) for 15 min at room temperature. The blocked membranes were then incubated overnight with primary antibodies, namely goat anti-SM22α (1:500; Abcam, Cambridge, UK), rabbit anti-OPN (1:1000; Abcam), and rabbit anti-MMP9 (1:1000; Abcam). After 12 h to 14 h, the membranes were incubated with a horseradish-peroxidase-conjugated secondary antibody (HuaBio, Hangzhou, China) for 1 h at room temperature. The immunoblots were probed using an enhanced chemiluminescence substrate (Thermo Fisher Scientific), and an imaging system (Bio-Rad Laboratories, Hercules, CA, USA) was used for blot detection and recoding of chemiluminescence. The results were normalized to that of GAPDH, and experiments were performed in triplicate.

Proteins were extracted from cells using RIPA cell lysis, and they were degenerated with 5× protein loading. The proteins were separated and transferred using sodium dodecyl sulfate polyacrylamide gel electrophoresis (Epizyme, China) and 0.2-μm polyvinylidene difluoride membranes (Millipore, USA). Subsequently, the membrane with proteins was incubated with primary and secondary antibodies as described in Supplementary Table 1.