Formation of a centrosomal Aki1–cohesin complex is required for centriole cohesion. (A) Immunoblots were performed on cytoplasmic, nuclear, and sucrose gradient fractions (centrosome preparations) from HeLa cells with the indicated antibodies. (B) HeLa cells were harvested 0 or 2 h after release from nocodazole arrest, and centrosomes were purified. Extracts obtained from indicated sucrose gradient fractions and chromatin fractions were immunoblotted with the indicated antibodies. Arrows indicate the full length (top) and C terminus (bottom) of Scc1. The top Scc1 blot shows a longer exposure of the bottom blot. (C) HeLa cells were transfected with control or Scc1 siRNA and costained for α-tubulin (α-tub; green) and centrin2 (red). Centrosomes are shown enlarged on the left. (D) Centrosomes were purified from thymidine (Thy)-arrested cells, nocodazole (Noc)-arrested cells, or cells released (Rel) from a nocodazole arrest for 2 h. Centrosomal proteins were immunoprecipitated with control rabbit IgG, anti-Aki1, or anti-SMC1 (for reciprocal immunoprecipitation [IP]) antibody. Immunoprecipitated proteins, nuclear/chromatin (Nu/ch) lysates, and centrosomal (Cen) lysates were immunoblotted with the indicated antibodies. (E) 293T cells were transfected with pFlag-CMV-2 vector encoding none (mock), WT-Aki1, or Aki1 mutants as indicated. Immunoprecipitated proteins and cell lysates were immunoblotted with antibodies to SMC1 or Flag. (F) Percentage of multipolar cells of mitotic cells in control or Aki1-2 siRNA–treated cells expressing AcGFP-mock, AcGFP-WT-rAki1, or AcGFP-ΔN415-rAki1. Error bars represent SD of triplicate experiments (150–200 total cells were scored per condition). (G) Centrosomes were purified from thymidine-arrested control cells, nocodazole-arrested control cells, or Aki1-depleted cells harvested by mitotic shake off. Cytoplasmic (C), nuclear (N), and centrosomal lysates (fractions 10–12) were immunoblotted with the indicated antibodies. Bars, 5 µm.