Ab7959

Cells were harvested and treated with lysis buffer on ice (KeyGEN, Nanjing, China), and a BCA kit (KeyGEN, Nanjing, China) was used to quantify protein concentration. Equal amounts of protein were loaded in SDS–PAGE gels. After separation in the gel, the protein was transferred on a PVDF membrane. Membranes were blocked in 2% BSA in TBS-T for 1 h, and then incubated overnight (4°C) with antibodies against CDCA2 (Abcam, ab209656 1:1000), p21 (santa cruz, sc-397 1:500), p27 (santa cruz, sc-528 1:200), cyclin D1 (CST, 2978 1:1000), cyclin E1 (Abcam, ab7959 1:200) or β-actin (Cell Signaling, 8H10D10 1:1000). After being washed in TBS-T, membranes were incubated with goat anti-rabbit HRP-conjugated secondary antibody (1:10,000; Abcam) or goat anti-mouse HRP-conjugated secondary antibody (1:10,000; Abcam) for 2 h at room temperature. The blots were visualized by ECL detection (Thermo Scientific). All experiments were repeated at least three times independently.

Whole cells were homogenized and treated with lysis buffer on ice (Kaiji, Nanjing, China), and a BCA kit (Kaiji, Nanjing, China) was used to quantify protein concentrations. Equal amounts of protein were loaded in SDS–PAGE gels. After separation in the gel, the protein was transferred onto a PVDF membrane. The membranes were blocked in 2% BSA in TBST for 1 h, and incubated overnight (4°C) with antibodies against ZYG11A (Abcam, ab177696 1:1000), p21 (santa cruz, sc-397 1:500), p27 (santa cruz, sc-528 1:200), Cyclin D1 (CST, 2978 1:1000), Cyclin E1 (abcam, ab7959 1:200) or beta-actin (Cell Signaling, 8H10D10 1:1000). After washing in TBST, the membrane was incubated with goat anti-rabbit HRP-conjugated secondary antibody (1:10,000; Abcam) or goat anti-mouse HRP-conjugated secondary antibody (1:10,000; Abcam) for 2 h at room temperature. The blots were visualized by ECL detection (Thermo Scientific), and all experiments were repeated triple times.