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Fig. 4. (A) Co-immunoprecipitation of AKAP450 with anti-RII{alpha} from Reh-RII{alpha} and Reh-RII{alpha}(T54E) cells in interphase and mitosis (obtained by double thymidine block). The upper panel shows immunoprecipitation of RIPA-buffer extracts of 200x106 cells using anti-RII{alpha} antibody. As a control, Reh cells were immunoprecipitated with anti-AKAP450 antibody. Samples were separated by 4.5% PAGE containing 2 M urea, transferred to nitrocellulose and detected using anti-AKAP450 antibodies. The AKAP450 band is indicated by an arrow. The lower panel shows the RII{alpha} content in the precipitated samples. (B) Immunospecificity of the affinity-purified polyclonal AKAP450 antibodies. Lane 1 contains purified centrosomes (untreated); lanes 2 and 3 contain Triton-X-100 insoluble and -soluble fractions from the centrosomal preparation in lane 1, respectively; lanes 4 and 5 contain RIPA-insoluble and -soluble centrosomal fractions, respectively. The blot was analyzed by RII overlay (lower panel) and western blotting using the affinity-purified polyclonal AKAP450 antibodies (upper panel).