Mouse monoclonal anti β actin

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).