Rabbit monoclonal anti mmp9

RIPA lysis buffer (Boster, Wuhan, China) was used to extract protein from indicated cells. BCA Protein Assay Kit (Thermo Scientific, USA) was used to measure protein concentration. A total of 60 μg of protein was separated on 10% SDS-PAGE gels and blotted onto 0.22 μm nitrocellulose membranes (Boster). The membranes were blocked for 2 hours with 5% nonfat dry milk diluted with tris‑buffered saline (TBS) and incubated with primary antibodies (rabbit monoclonal anti-OLFM4 (1:2,000), rabbit monoclonal anti-phosphorylated-FAK antibody (1:500), rabbit monoclonal anti-FAK antibody (1:1,000), rabbit monoclonal anti-MMP2 (1:1,000), rabbit monoclonal anti-MMP9 (1:3,000), and mouse monoclonal anti-β-actin (1:3,000) (Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4℃. The membranes were washed with tris-buffered saline containing 0.1% Tween20 (TBST), and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit, 1:3,000; goat anti‑mouse, 1:2,000; Wuhan Boster) for 1 h at 37℃. Enhanced chemiluminescence reagent (Merck Millipore, Germany) was used to detect the signal on the membrane. The data was analyzed via densitometry using Image-Pro plus 6.0 software (Media Cybernetics, Rockville, MD, USA) and normalized to the expression of the internal control (β-actin).

Mouse monoclonal antibodies against CDH1 (#610181) and STAT3 (#610189) were obtained from BD Transduction Laboratories. Rabbit monoclonal anti-MMP-9 (#13667) and rabbit polyclonal anti-phosphor-STAT3 (#9131) antibodies were obtained from Cell Signaling Technology. Mouse anti-β-actin monoclonal antibody (#TA-09) and the rabbit monoclonal anti-CDH1 (#ZA-0565) and anti-MMP-9 (#ZA-0562) antibodies used in the IHC assay were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. Purified AG490 powder (#T3434) was purchased from Sigma Aldrich.