(A) Growth factor regulation of cyclin E-associated kinase activity following p27 immunodepletion. Sparsely seeded cells were treated with TGF-β (100 pM) and HGF (220 pM), as indicated, followed by lysis in NP-40 buffer. Lysates were immunoprecipitated with cyclin E antibody (lanes 1 to 4) or with three subsequent cycles of polyclonal p27 antibody, followed by cyclin E immunoprecipitation (lanes 5 to 8). Kinase assays towards histone H1 were carried out as described in Materials and Methods. Relative kinase activities after subtraction of the kinase activity given by nonimmune serum compared to control cells (set at 100) are indicated. (B) Effects of p27 immunodepletion steps on cyclin E, Cdk2, p27, and their complexes. In parallel with experiment represented by panel A, cell lysates before and after three immunodepletion cycles with anti-p27 antibody were analyzed by immunoprecipitation (IP) with the indicated antibody, followed by immunoblotting (WB). Total cyclin E and p27 were analyzed by immunoblotting. Fold changes compared to controls are shown below each analysis.