FIG. 3.

pRb changes the phosphorylation state of Py T Ag in extracts of FM3A cells. (A) Py T Ag (0.6 μg) was incubated in complete Py ori-DNA replication reaction mixtures at 33°C for 3 h in the presence (+) or absence (−) of pRb (1.2 μg). Two different preparations of FM3A extract (1 and 2, 300 μg each) were used. The total reaction volume (50 μl) was analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with Py T Ag-specific antibody PAb F5. (B) Py T Ag (0.6 μg) was incubated in a complete replication reaction including [γ-³²P]ATP at 33°C for 3 h with (+) or without (−) pRb (1.2 μg). Py T Ag was immunoprecipitated with Py T Ag-specific antibody PAb F5 and analyzed by SDS-PAGE. The Western blot (Western) was probed with PAb F5 and IF-8 (specific for pRb) before being subjected to autoradiography (Autorad). Molecular masses of marker proteins are indicated in kilodaltons on the left; the positions of Py T Ag and pRb are indicated on the right.