Ab70350

RIPA lysis buffer was used to extract the total proteins from the cells, and the protein concentration was quantified using a BCA kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. A total of 30 µg protein/lane was loaded on a 10% SDS-gel, resolved using SDS-PAGE and transferred to a PVDF (Roche Diagnostics GmbH). The membranes were blocked with 5% nonfat milk for 1 h at room temperature, and then incubated with the corresponding primary antibodies anti-NCAPG (1:800, ab70350, Abcam), anti-Bcl-2 (1:1,000, ab32124, Abcam), anti-Bax (1:1,000, ab182733, Abcam), anti-Bad (1:1,000, ab32445, Abcam), anti-MMP2 (1:1,000, ab92536, Abcam), anti-MMP9 (1:1,000, ab76003, Abcam), anti-p-EGFG (1:1,000, ab40815, Abcam), anti-EGFR (1:1,000, ab52894 Abcam), anti-p-JAK1 (1:1,000, ab138005, Abcam), anti-JAK1 (1:800, ab133666, Abcam), anti-STAT3 (1:1,000, ab68153, Abcam), anti-p-STAT3 (1:1,000, ab76315, Abcam), anti-GAPDH (1:1,000, ab9485, Abcam) overnight at 4˚C. The following day, the membranes were incubated with goat anti-rabbit IgG H&L (HRP)-conjugated secondary antibodies (1:5,000, ab7090, Abcam) for 1 h at room temperature. The expression levels of the different proteins were detected using enhanced chemiluminescence reagent (Bio-Rad Laboratories, Inc.). The data were analyzed using ImageJ version 1.46 (National Institutes of Health).