Anti mmp 9

The fibroblasts were lysed in lysis buffer (Beyotime) and centrifuged for supernatant recovery. The protein concentrations were determined using a BCA kit (Beyotime). After being separated by 10% SDS-PAGE, the proteins (20 µg) were transferred onto nitrocellulose membranes (Millipore) and blocked with 5% non-fat dry milk for 1 h at room temperature. The membranes were then first incubated at 4°C overnight with anti-PTEN (1:200; cat. no. 9188), anti-DNMT1 (1:1,000; cat. no. 5032), anti-DNMT3a (1:1,000; cat. no. 49768), anti-DNMT3b (1:1,000; cat. no. 48488), anti-PI3K (1:1,000; cat. no. 4255), anti-p-PI3K (1:1,000; cat. no. 4228), anti-Akt (1:1,000; cat. no. 4685), anti-p-Akt (1:1,000; cat. no. 4056), anti-MMP-2 (1:1,000; cat. no. 87809) (all from Cell Signaling Technology), anti-α-SMA (1:350; cat. no. A03744, BosterBio), anti-MMP-9 (1:500; sc-21733), anti-caspase-3 (1:200; sc-271759), anti-collagen I (1:1,000; sc-376350), anti-collagen III (1:1,000; sc-271249) and anti-β-actin (1:1,000; sc-58673) (all from Santa Cruz Technology) antibodies and then with secondary antibodies [goat anti-rabbit IgG H&L (HRP) (1:5,000; ab6721, Abcam)] for 2 h at room temperature. The proteins were visualized using an ECL kit (Amersham Biosciences, Germany), and analyzed with the Bio-Rad ChemiDoc™ XRS+ System and Image Lab™ Software version 4.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cells were collected and lysed with cell lysis buffer for western blotting (Beyotime, Haimen, China). The proteins (30 μg per lane) were separated on 12% SDS-polyacrylamide gels and transferred on a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Immunoblotting of the membrane was performed using the following primary antibodies: anti-CDK6 (Boster, Wuhan, China), anti-cyclin D1 (Affinity, Cincinnati, OH, USA), anti-MMP-2 (Boster), anti-MMP-9 (Boster) and anti-β-actin (4 A Biotech, Beijing, China). The signals were revealed after incubation with the recommended secondary antibodies using an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA). β-actin was used as the control.