Er30607

Briefly, protein was extracted from the cells using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology). Sample protein concentrations were quantitated using the BCA method and 30 µg total protein per lane were separated by 10% SDS-PAGE and then transferred onto PVDF membranes. The PVDF membranes were blocked with 5% BSA for 2 h and then incubated with primary anti-human antibodies against NKRF (ab168829, at 1:1,000 dilution; Abcam), MMP-9 (EM1801-22, at 1:500 dilution, HuaBio Inc.), MMP-2 (ER40806, at 1:1,000 dilution, HuaBio Inc.), VEGF (ER30607, at 1:1,000 dilution, HuaBio Inc.), NF-κB-p65 (D14E12, #8242, at 1:2,000 dilution, Cell Signaling Technology, Inc.) and GAPDH (M1310-2, at 1:3,000 dilution; HuaBio Inc.) overnight at 4°C. The membranes were incubated with the corresponding horseradish peroxidase (HRP)-labeled goat anti-rabbit (A0208, at 1:10,000 dilution; Beyotime Institute of Biotechnology Inc.) or anti-mouse IgG antibody (A0216, at 1:10,000 dilution; Beyotime Institute of Biotechnology Inc.) for 1 h at room temperature. After washing, the WB signal was detected using an enhanced chemiluminescence system (Bio-Rad Laboratories, Inc.) and the densitometric analysis of the images was performed using Image Lab 6.0.1 software (Bio-Rad Laboratories, Inc.).