Anti mmp12

For preparation of protein extracts, 12 pairs of cancer and adjacent normal tissues were crushed with a mortar under ice cold conditions and lysed with RIPA lysis buffer together with protease inhibitors. Cells were collected and lysed with RIPA lysis buffer together with protease inhibitors. After centrifugation at 12,000 rpm at 4°C for 20 min, supernatants were collected and protein concentration was determined using the Pierce™ BCA protein assay (Thermo, Waltham, MA, USA). Proteins were separated by electrophoresis on a 10% SDS-polyacrylamide gel, electroblotted onto a PVDF membrane, and blocked by 5% nonfat dry milk for 1 h. Membranes were then washed in TBST three times for 5 min and then incubated with anti-MMP1 (Abcam, Cambridge, MA, USA), anti-MMP2 (Abcam), anti-MMP3 (Abcam, USA), anti-MMP7 (Abcam, USA), anti-MMP8 (Abcam, USA), anti-MMP9 (Invitrogen, Waltham, MA, USA), anti-MMP11 (Bioss, Beijing, China), anti-MMP12 (Abcam, USA), anti-MMP14 (Abcam, USA), anti-MMP17 (Abcam, USA), anti-MMP19 (Bioss, China), anti-MMP28 (Abcam, USA), anti-Collagen (Abcam), anti-TIMP2 (Bioss, China), or anti-GAPDH (Abcam). Subsequently, the membranes were washed with PBST and incubated for 1 h with goat anti-rabbit IgG (Abcam). Finally, membranes were washed three times and immunoreactivity was determined by using a Chemi DOC™ XRS+ system (BioRad Laboratories, Hercules, CA, USA).

Total protein was extracted from cell lysates after cells were harvested and centrifuged at 4°C and 16,000 × g for 20 min. Protein concentrations were measured by BCA method (Thermo Fisher Scientific, Inc.). Subsequently, proteins were separated via 10% SDS-PAGE for 2 h and transferred onto PVDF membranes by means of a wet transfer electrophoresis tank (Bio-Rad Laboratories, Inc.) for 2 h. Sealed by 5% skim milk for 1 h at room temperature, the membranes were then incubated overnight at 4°C with the following primary antibodies: anti-SIRT6 (1:2,000; cat. no. ab191385; Abcam); anti-cleaved-caspase-3 (Asp175; 1:2,000; cat. no. 9664; Cell Signaling Technology, Inc.); anti-p53 (1:10,000; cat. no. ab32389; Abcam); anti-MMP12 (1:1,000; cat. no. ab52897; Abcam); anti-MMP9 (1:1,000; cat. no. Ab76003; Abcam); anti-GAPDH (1:2,000; cat. no. ab181602; Abcam). After being washed with PBS for twice, the membranes were further incubated with horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. ab6721; Abcam). Visualization of bands was conducted by electrochemiluminescence substrate (Thermo Fisher Scientific, Inc.).