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Fig. 7. Western blotting of in vitro kinase assays with crude cell extracts. Lysates from NGF/p25-expressing cells (6 hours of tetracycline treatment) were used for Rb-C fusion protein phosphorylation, alone and in the presence of the kinase inhibitors roscovitine, PD98059, SB203580 and SP600125. As negative control, an in vitro kinase assay was performed with lysate from NGF-mock cells treated for 6 hours with tetracycline. Phosphorylation of Rb-C fusion protein (P-Rb) was monitored by P-Rb Ser807/811 and Ser795 antibodies, which showed similar data. After stripping, membranes were probed with a phosphorylation-independent antibody against Rb (C-15) to evaluate the amount of loaded Rb-C fusion protein (Rb). The lower side of membrane was analysed by immunoblotting for the levels of p25 and Cdk5, which attests to the presence of equivalent amounts of crude lysate in each kinase reaction.
