Quantitative Western Blot Analysis of Vascular Protein Expression

Western blotting experiments were performed as previously described [30 (link)]. Briefly, total protein of VSMCs and vascular tissue was extracted with RIPA lysis buffer (Beyotime, Shanghai, China) and protease inhibitor (Thermo Fisher Scientific, CA, USA) mixture. The protein concentration was measured using a BCA kit (Thermo Fisher Scientific, CA, USA). Equal amounts of protein were separated by 12.5% SDS-PAGE and then transferred to PVDF membrane (MilliporeSigma, MA, USA), blocked in 5% milk for 1 h at room temperature. Then, they were incubated with the following primary antibodies at 4 °C overnight: anti-SLC6A6 (1:1000; Cat#ab236898; Abcam, MA, USA), anti-α-SMA (1:10,000; Cat#55135-1-AP; Proteintech, Wuhan, China), anti-SM22α (1:10,000; Cat# ab14106; Abcam), anti-SMMHC (1:1000; Cat#21404-1AP; Proteintech), anti-CNN1 (1:1000; Cat#17819; Cell Signaling Technology, MA, USA), anti-cyclin D1 (1:10000; Cat#ab134175; Abcam, MA, USA), anti-β-catenin (1:1000; Cat# ab32572; Abcam, MA, USA), and anti-β-actin (1:1000; Cat# 66009-1-Ig; Proteintech,Wuhan, China). The membranes were then incubated with anti-mouse or anti-rabbit HRP-conjugated antibodies (1:10,000; EasyBio, Beijing, China). Images were acquired using an optical scanner and analyzed using ImageJ software ( ImageJ software v1.6.0, MD, USA) [31 (link)].