Ab3235

Total protein was prepared in RIPA lysis buffer (Beyotime, Nantong, China) on ice, then extracted by 12% SDS-PAGE and shifted to PVDF membranes (Millipore, Billerica, MA). Membranes were sealed for 1 h with 5% skim milk and incubated with primary antibodies including anti-GAPDH (ab9485, abcam), anti-Bax (ab32503, abcam), anti-Bcl-2 (ab32124, abcam), anti-caspase 3 (ab3235, abcam), anti-cleaved caspase 3 (ab2302, abcam), anti-CDCA4 (ab227953, abcam), anti-ki-67 (ab270650, abcam), anti-PCNA (ab29, abcam), and anti-EGR1 (ab194357, abcam) respectively all night at 4 °C. After being washed in TBST, secondary antibodies tagged with HRP were added and incubated for 1 h at 37 °C. The protein bands were quantified by ECL Prime Western Blotting Detection reagent (GE Healthcare, Chicago, IL).

Cells were lysed in RIPA buffer (Beyotime, China) with freshly added 0.01% protease inhibitors (Roche) and then centrifuged at 15,000 × g at 4 °C for 15 min. The supernatants were harvested and quantified using the BCA protein assay kit (Beyotime, China) according to the manufacturer’s instructions. Then 20 μg total proteins were applied and resolved on 4–12% SDS-PAGE gradient gels and transferred to 0.22 µm PVDF membranes. Membranes were blocked with 5% nonfat milk and then incubated with the following primary antibodies: caspase-3 (Abcam, ab3235, 1:1000), Cleaved Caspase-3 (Abcam, ab32042, 1:500), OVOL2 (Abcam, ab1-69469, 1:500), and β-actin (Abcam, ab8226, 1:500). The membranes were incubated at 4 °C overnight and then washed by TBST solution for three times. After that, they were applied with appropriate secondary antibodies. Signals were detected using electrochemiluminescence methodology. All experiments were repeated at least three times.