Fig. 7. Co-immunoprecipitation of pRB with E2F-1 and E2F-4 antibodies. Reh cells were treated with forskolin (100 µM) for the hours indicated. (A) Total cell lysate (500 µg) from treated or untreated cells were subjected to immunoprecipitation as described in Materials and Methods, using antibodies specific for E2F-1 or E2F-4 as indicated. The E2F-complexes were resolved on a 10% SDS-PAGE and the resulting immunoblots were detected with antibodies specific for pRB. (B) 30 µg of the same cell lysates were subjected to immunoblot analysis to detect the total amount of E2F-1 or E2F-4 as indicated. One representative experiment of three is shown.