Human CRL2^ZYG11A/B negatively regulates the level of cyclin B1 in mitosis. (A) Western blot demonstrating the effectiveness of ZYG11A and ZYG11B siRNA knockdown of FLAG-ZYG11A and FLAG-ZYG11B expressed in U2OS cells. The relative FLAG-ZYG11A/B to actin ratio is given below the blots. Similar results were obtained in two independent experiments. (B) IP/Western blot with anti-ZYG11A and anti-ZYG11B antibodies demonstrating the effectiveness of the siRNA knockdown of the endogenous proteins in U2OS cells. The relative ZYG11A/B to actin ratio is given below the blots. Comparable RNAi knockdowns were also observed in HEK293T cells (not depicted). (C) ZYG11A, ZYG11B, or ZYG11A/B siRNA knockdowns increase the level of endogenous cyclin B1 in cells treated with proTAME at 2 h after release from RO-3306 G₂ arrest. Epifluorescence images of representative β-tubulin, DNA, and endogenous cyclin B1 staining of metaphase-arrested proTAME-treated cells. Bar, 10 µm. (D) Bar graph shows the relative level of cyclin B1 ± SEM in the indicated siRNA treatments; n = 20 cells for each condition. Bar graphs presenting the ratios of the chromosome-to-cytoplasm (E) and chromosome-to-spindle (F) immunofluorescence signals ± SEM for endogenous cyclin B1 for the experiment described in C; n = 25 cells for each condition. For C–F, similar results were obtained in two independent experiments. ***, P < 0.001; ****, P <0.0001.