Mmp12

Specific antibodies (rabbit anti-human antibodies) to MST4, FAK, MMP12, JUN, and GAPDH were provided by Cell Signaling Technology (Beverly, MA, USA) and Abcam (Cambridge, MA, USA). Infrared-labeled secondary antibodies (goat anti-rabbit antibodies) to IRDye 800 were obtained from Li-Cor Biosciences (Lincoln, NE, USA). Cells were washed twice with PBS and lysed in a cell lysis buffer for the reagent of Western blotting and immunoprecipitation (Beyotime, Beijing, China) and protease inhibitor phenylmethanesulfonyl fluoride (Beyotime). Equal amounts of protein were heated to 100°C for 5 minutes with Laemmli sample buffer, and then protein samples were separated using 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were incubated with the primary antibodies (1:1,000) at 4°C overnight, washed three times in PBST, and incubated with peroxidase-conjugated secondary antibodies (1:10,000) for 1 hour at 25°C. The membranes were scanned and the net intensities of the bands were quantified using Odyssey Software Version 3.0 system (Li-Cor Biosciences). Then, the protein expression levels of each gene were measured with GAPDH protein as a reference at membrane.

To evaluate the expression of MMPs, western blot analysis was performed. The cells were lysed with PRO-PREP™ solution (Intron Biotechnology, Sungnam, Korea), and equal amounts of cell extracts were analyzed using 4% to 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) and blocked with 4% nonfat milk in Tris-buffered saline and 0.1% Tween 20 at room temperature. Primary antibodies (MMP 1–2: Cell Signaling, Beverly, MA, USA; MMP9: Santa Cruz Biotechnology), secondary antibodies (Enzo Life Sciences, Minneapolis, MN, USA), and Amersham ECL Select Western blot detection reagent (GE Healthcare Life Sciences, Buckinghamshire, UK) were used. Anti-ACTB antibody (Sigma-Aldrich) was used for each probing. ImageJ software (version 1.52a; National Institutes of Health, Bethesda, MD, USA) was used for band intensity quantification. In order to calculate relative protein expression ratios, the protein expressions in the treated cells were divided by those of the control cells.