Western blot analysis

Total protein was extracted from cells with a radio-immunoprecipitation assay buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) containing PMSF, incubated on ice for 30 min and centrifuged at 4°C for 10 min (12,000 × g). The supernatant was obtained. The protein concentration of the supernatant of each sample was determined using a bicinchoninic acid kit (cat. no. 23225; Thermo Fisher Scientific, Inc.), and deionized water was then used to adjust the amount of protein. Next, a 10% SDS-PAGE gel (cat. no. P0012A; Beyotime Institute of Biotechnology, Shanghai, China) was prepared and 50 µg of the protein sample was added to each well. Electrophoresis was conducted at a constant voltage of 80 V for 2 h. The proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (cat. no. ISEQ00010; EMD Millipore, Billerica, MA, USA) with a voltage of 110 V for 2 h. The PVDF membrane was blocked with TBST buffer containing 5% skimmed milk powder for 2 h. Then, the membrane was washed with TBST and incubated with rabbit polyclonal antibody Col I (1:100; cat. no. ab34710), rabbit polyclonal antibody Col III (1:100; cat. no. ab7778), and β-actin antibody (1:100; cat. no. ab8224; all Abcam) at 4°C overnight, followed by three washed with TBST, 10 min each time. The membrane was then washed with 0.1% PBS/Tween-20 (PBST) 3 times at room temperature, 10 min each time, immersed in enhanced chemiluminescence reaction solution (cat. no. WBKLS0100; EMD Millipore), and developed. β-actin was used as the internal reference, and the ratio of the gray values of the target protein band to the internal reference band was used as the relative protein expression and analyzed by Quantity One v4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).