AKAP450 is required for centrosome cohesion. (A) Western blots (wb) showing siRNA-mediated depletion of CDK5RAP2 (CDK5R2) and AKAP450 (akap) in HeLa cells. Depletion of CDK5RAP2 does not interfere with the localization of AKAP450 to the Golgi and the centrosome during interphase, but it prevents its accumulation in the mitotic centrosome (arrowheads). Depletion of AKAP450 does not prevent the localization of CDK5RAP2 to the centrosome. Note that the Golgi disperses in AKAP450-depleted cells (Larocca et al., 2004), so CDK5RAP2 cannot accumulate in the Golgi. Blue, DNA; red, CDK5RAP2; green, AKAP450. Bar, 10 µm. (B) Graph shows quantification of centrosome splitting in mock and specific siRNA-treated cells. n = 3; at least 150 cells per experiment. (C) Centrosomes split in cnn1^−/− DT40 cells. Anti-CDK5RAP2 antibody does not stain the centrosome in interphase cnn1^−/− cells, but centrosomal γ-tubulin signal is comparable between wt and cnn1^−/− centrosomes. Insets show close ups of normal (wt) and split (cnn1^−/−) centrosomes. Blue, DNA; red, CDK5RAP2; green, γ-tubulin. Arrow marks the identity of the cell in inset. Bar, 5 µm. Graph shows quantification of centrosome splitting in wt and mutant cells (n = 4; 150 cells per experiment). P-values were calculated by two-tailed unpaired Student’s t test. Error bars represent SD.