Protease inhibitors

The kit used in protein lysis was radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China), which was added with protease inhibitors (Bioss, Beijing, China). The kit used in concentration examination of proteins was the BCA kit (Thermo Fisher Scientific, Inc.). Proteins of equal quantity (30 μg) were separated by 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The samples were then mounted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). Then, an overnight incubation was performed on 5% skimmed milk powder-blocked membranes (1 h) as well as primary antibodies. The used antibodies were PRR11 (ab237526, Abcam, UK) and GAPDH (ab9485, Abcam, UK). Following 3 washes (10 min/time) with PBST, the treated membranes were subjected to 1 h of secondary antibody incubation (ab6721, Abcam, UK, goat anti-rabbit IgG, horseradish-labeled). Following 3 washes with PBST (10 min/time), the membranes were scanned under an optical illuminator (GE, USA).

Total proteins from the cells were lysed in radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China, cat. no.: P0013B) containing protease inhibitors (BIOSS, Beijing, China). Protein concentration was determined using a BCA Protein Assay kit (Thermo Scientific Pierce, U.S.A., Cat. lot.: 23225). An equal amount of total proteins was then separated by 10% SDS/PAGE and transferred on to a polyvinylidene difluoride membrane (cat. no. PK-NEF1002; PerkinElmer, Inc., Boston, MA, U.S.A.). Next, fat-free milk (5%) was used to block the membranes for 2 h at room temperature. Following blocking, the membranes were incubated with primary antibodies overnight at 4°C. The blots were subsequently incubated with horseradish peroxidase–conjugated secondary antibodies (Wuhan Boster Biological Technology, Ltd., Wuhan, China; cat. no. BA1054) and then developed using enhanced chemiluminescence detection kit (32106, Thermo Scientific™ Pierce, U.S.A.) according to the manufacturer’s protocol. The primary antibodies used included anti-β-actin (4967, CST, U.S.A.), Col I (ab90395, Abcam, U.S.A.), anti-Col III (ab23445, Abcam, U.S.A.), anti-S100A4 (ab41532, Abcam, U.S.A.), anti-CXCR4 (ab219178, Abcam, U.S.A.), and anti-ALP (ab83259, Abcam, U.S.A.) antibodies.