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Fig. 6. Immunoprecipitation and in vitro kinase assays. Lysates from NGF-mock and NGF-p25 cells, both treated for 6 hours with tetracycline, were subjected to immunoprecipitation (IP) with the indicated antibodies. (A) Western-blot analyses. Following in vitro kinase assays, Rb-C fusion protein (68 kDa) phosphorylation was evaluated by immunoblotting using P-Rb Ser807/811 and P-Rb Ser795 antibodies that give similar results (representative immunoblot with P-Rb Ser807/811 is shown). To evaluate the amount of loaded Rb-C fusion protein, blots were stripped and reprobed with a phosphorylation-independent antibody (C-15) directed against the C terminus of Rb (Rb). The lower sections of blots were also probed with specific antibodies to check the presence of the following proteins: p25, Cdk5, Cdk2, Cdk4 and Cdk6. (B) In vitro kinase assays in the presence of (-³²P)ATP. Arrowhead indicates Rb-C fusion protein with incorporated ³²P, which is only seen in in vitro kinase assay with purified p25-Cdk5 complex.

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