Anti mmp 9

The experimental cells of the same growth time and good condition were collected. After washing with ice‐cold PBS, cells were added to the RIPA lysis buffer for 20 minutes. After centrifuged (13 188 g) with 4°C, protein was determined using a BCA protein assay kit. Western blotting was carried out according to standard protocol. The positive stripe was analyzed by Gel Pro 4.0 optical density analysis software (Media Cybernetics, Rockville, MD, USA), and the accumulated optical density reference value of integrated optical density was measured. The following antibodies were used in this study: anti‐INHBB (ab69286; Abcam, Cambridge, UK), anti‐E‐cadherin (YT1454; ImmunoWay, Plano, TX, USA), anti‐vimentin (YT4880; ImmunoWay), anti‐VEGF‐A (YT5108; ImmunoWay), anti‐MMP‐9 (YT1892; ImmunoWay), anti‐Smad2/3 (YT4332; ImmunoWay), anti‐Smad4 (YT4337; ImmunoWay), anti‐p‐Smad2/3(T8) (YP0362; ImmunoWay), anti‐TGF‐β1 (YT4632; ImmunoWay), anti‐β‐actin (20270; ProMab, Richmond, CA, USA), anti‐p53 (10442‐1‐AP; Proteintech, Rosemont, IL, USA).

Total protein was extracted from cell lysates using radioimmunoprecipitation assay buffer (Beyotime Biotechnology, Shanghai, China). Equal amounts of total protein samples were separated by SDS-polyacrylamide gel electrophoresis and electrotransferred from the gel to polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA). The membranes were blocked with 5% fat-free milk or bovine serum albumin, and then immunoblotted using the following primary antibodies: rabbit anti-PGAM1 (1:1000; Abcam), rabbit polyclonal anti-cleaved caspase-3 (1:500, #9664; Cell Signaling Technology, Danvers, MA, USA); and rabbit polyclonal anti-Bcl-2, anti-Bax, anti-matrix metallopeptidase (MMP)-2, and anti-MMP-9 (all 1:500; all Immunoway, Plano, TX, USA). Anti-β-actin staining (1:1000; Bioworld Technology, Louis Park, MN, USA) was used as an internal control. Finally, the membranes were incubated with the appropriate secondary antibodies (1:5000; Boster Ltd., Wuhan, China). Signals were visualized using an enhanced chemiluminescence detection system (Pierce Biotechnology, Rockford, IL, USA) in accordance with the manufacturer's instructions.