Mmp9 13667

Western blotting assay was conducted as we have previously reported⁴⁹ . The primary antibodies included MMP2 (A6247), STAT3 (A11216) (Abclonal, Wuhan, China), p-STAT3 (9145), GAPDH (5174), iNOS (39898), MMP9 (13667) (Cell Signaling Technology, CST, Boston, MA, USA), β-catenin (17565-1-AP), β-actin (20536-1-AP), E-cadherin (20874-1-AP), N-cadherin (22018-1-AP), Vimentin (10366-1-AP) (Proteintech Group, Chicago, IL, USA), CCR5 (AF6339), CCL5 (AF5151), Arg1 (DF6657) (Affinity Biosciences, Cincinnati, OH, USA).

Total protein was isolated using RIPA lysis buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride membranes. The membrane was and probed with diluted primary rabbit antibodies against Caspase-3 (ab13847), Bcl-2-associated X protein (Bax; ab32503), B-cell lymphoma 2 (Bcl-2) (ab32124), Bcl-XL (ab32370), TGF-β1 (ab92486), β-actin (ab8227), GAPDH (ab181602), E-cadherin (3195), N-cadherin (13116), matrix metalloproteinase (MMP)3 (14351), MMP9 (13667), Smad2 (5339), Smad3 (9523), phosphorylated (p)-Smad2 (18338), and p-Smad3 (9520, Cell Signaling Technologies) overnight at 4°C. The antibodies were from Abcam except for p-Smad2/3. After washing, membrane was re-probed with the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ab205719, 1: 2000, Abcam) for 1 h. The protein bands were visualized using enhanced chemiluminescence (EMD Millipore). β-actin and GAPDH were used as internal controls. The gray values were analyzed with Image J software.