The BTB proteins KLHL21 and KLHL22 interact with Cul3 and regulate mitosis. (A) Immunoprecipitation of human KLHL9 and KLHL13 was analyzed by mass spectrometry, and associated Cul3 (circle) and BTB proteins (boxes) are shown (dark gray). Immunoprecipitation of Cul3 identified KLHL12, KLHL22, and the highly homologous KLHL9 and KLHL13 and BTBD1 and BTBD2 (light gray). (B) HeLa extract was immunoprecipitated with control (anti-IgG) or anti-KLHL21 antibodies. Input, supernatants (Sup), and eluates (IP) were analyzed by immunoblotting. (C) HeLa cells expressing H2B-mRFP and mEGFP–α-tubulin were treated with the indicated siRNAs for 48 h and analyzed by live video microscopy. (D) Quantification of the phenotype in C. The percentage of cells with a delay from prophase to anaphase (>50 min) was quantified (n = 50). (E) HeLa cells were treated with the indicated siRNAs for 72 h, and the percentage of multinucleated cells was quantified (n = 500). Example images are shown in Fig. S1 D. (D and E) Bars represent the mean with standard deviation of three independent experiments. Bars, 10 µm.