E8651

Total protein in cells was extracted using the cell lysis buffer (R0278, Sigma‐Aldrich, Merck KGaA), and the protein concentration was examined by the bicinchoninic acid kit (BCA1, Sigma‐Aldrich). The protein was separated by SDS‐PAGE and wet‐transferred to polyvinylidene fluoride membranes, which were blocked by 3% nonfat milk for 45 min and probed by the antibodies of E2F3 (1:500, E8651, Sigma‐Aldrich), PRC1 (1:1000, 15617‐1‐AP, Proteintech Group, Inc.), BIRC5 (1:1000, #2802, Cell Signaling Technology), and GAPDH (1:1000, #97166, Cell Signaling Technology) overnight at 4°C. The next day, the membranes were incubated with goat anti‐mouse IgG (HRP; 1:2000, ab6789, Abcam Inc.) at 20–25°C for 1 h. The protein blots were developed by enhanced chemiluminescence (WBULS0100, Sigma‐Aldrich), and the level of target protein relative to the internal loading (GAPDH) was analyzed.

According to the protocol of the EZ‐Magna ChIP® A kit (17‐409, Sigma‐Aldrich), the HNE3 cells were treated with formaldehyde and then with glycine to terminate the protein‐DNA cross‐linking. After that, the cells were lysed and ultra‐sonicated to truncate DNA. The lysates were incubated with anti‐E2F3 (1:50, E8651, Sigma‐Aldrich) or isotype control mouse IgG at 4°C overnight for immunoprecipitation. The protein–DNA complexes were eluted and de‐crosslinked by proteinase K. The DNA was collected and purified, in which the expression of PRC1 and BIRC5 promoter fragments was analyzed by qPCR.