Tissue Protein Extraction and Analysis

The total protein extraction from tissues or cells was conducted with radioimmunoprecipitation assay (RIPA) lysis buffer (P0013c, Beyotime Biotechnology, Shanghai, China) containing phenyhnethylsulfonyl fluoride (PMSF). The supernatant was incubated on ice for 30 min, and centrifuged at 4 °C and 8000 g for 10 min, followed by bicinchoninic acid (BCA) protein assay (CWBio, Beijing, China). The extracted proteins were stored at – 80 ℃ after packaging. The proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for 1 h, transferred onto nitrocellulose membranes, and then blocked with 5% skimmed milk (PBS) for 2 h. The membranes were incubated for 30 min at ambient temperature with rabbit polyclonal antibodies MMP11 (1:1000, ab119284), Bcl-2 (1:1000, ab182858), Cyclin D1 (1:1000, ab40754), CDK4 (1:1000, ab199728), Bax (1:1000, ab182733), and GAPDH (ab9485, 1:2500) (Abcam, Cambridge, UK). HRP-coupled antibody to immunoglobulin G (IgG) (A21020, 1:5000, AmyJet Scientific Inc., Wuhan, China) was loaded for 1-h incubation. The immunoblots were subsequently developed using enhanced chemiluminescence reagent (BB-3501, Amersham Biosciences, Piscataway, NJ), and imaged on the gel imager. An imaging system (Bio-Rad Inc., Hercules, CA) was utilized for gray value analysis with the application of Quantity One v4.6.2 software [24 (link)].