CDK5RAP2 interacts with and targets AKAP450 to the centrosome. (A) CDK5RAP2 colocalizes with AKAP450 in HeLa cells. Blue, DNA; red, AKAP450; green, CDK5RAP2. Bar, 10 µm. (B) CDK5RAP2 coimmunoprecipitates with AKAP450 from HeLa cell extracts. A rabbit IgG mix was used as negative control. Input lane shows cytoplasmic extract before immunoprecipitation (IP). Input and supernatant correspond to 10% of the pellet. CDK5RAP2 antibody immunoprecipitated 26% of total CDK5RAP2 and 40% of total AKAP450 protein. The extent of CDK5RAP2 immunoprecipitation may be underestimated because the CDK5RAP2 signal is saturated. SN, supernatant; P, pellet. (C) Western blots (wb) of wt, cnn1^lox, cnn2^−/−, tagAKAP-wt tagAKAP-cnn1^lox, and tagAKAP-cnn2^−/− cell extracts. α-Tubulin serves as the loading control. (D) Anti–protein G antibody stains the mitotic centrosomes of tagAKAP-wt but not of tagAKAP-cnn1^lox and tagAKAP-cnn2^−/− cells. Blue, DNA; red, protein G (AKAP450); green, γ-tubulin. Bar, 5 µm.