KLHL21 regulates midzone recruitment of Mklp2 but not PRC1 or anillin. (A–C) HeLa cells treated with control or KLHL21 siRNA were analyzed by IF for PRC1 (A), anillin (B), or Mklp2 (C) and Aurora B. DNA was stained with DAPI. (D and F) Quantification of the relative intensities of PRC1 (D) and Mklp2 (F). The intensity on the midzone was divided by the intensity in the cytoplasm (n = 20). (E) Percentage of anaphase cells with anillin recruitment to the cleavage furrow (n = 50). In KLHL21 siRNA, only cells with mislocalized Aurora B were included. (D–F) Bars represent the mean with standard deviation of three independent experiments. (G) HeLa cells were treated with the indicated siRNAs for 72 h, and extracts were analyzed by immunoblotting. α-Tubulin was used as a loading control. (H) Extracts prepared from cells treated with control siRNA, siRNA depleting KLHL21, or siRNA depleting both KLHL9 and KLHL13 were immunoprecipitated with anti-Cul3 antibodies. Inputs and eluates (Cul3 IP) were analyzed by immunoblotting. Bars, 5 µm.