Fig. 5. Inhibitors of phosphatase 1 and 2A prevent forskolin-mediated dephosphorylation of pRB. Reh cells were treated for 4 hours with forskolin (100 µM), in the presence or absence of tautomycin (10 µM). Total cell lysate (50 µg) from treated and untreated cells were subjected to immunoblot analysis (as described in Materials and Methods), using antibodies specific for (A) pRB, or (C) cyclins as indicated. (B) For analysis of kinase activity, 500 µg of total cell lysate was subjected to immunoprecipitation (IP) with the appropriate antibodies, and kinase assays were performed, as described in Materials and Methods, using histone 1 or GST-RB as substrate. One representative experiment of three is shown.