Cdk2- and cyclin E-associated kinase activities of TGF-β- and HGF-treated Mv1Lu cells. Cells were treated with the indicated growth factors, followed by lysis in NP-40 buffer. Immunoprecipitations with the indicated antibodies and kinase assays were carried out as described in Materials and Methods. (A) Cdk2-associated kinase activity with histone H1 as a substrate. (B) Cdk2 (lanes 1 to 4)- and cyclin E (lanes 5 to 8)-associated kinase activities with GST-Rb as a substrate. The figures show relative kinase activities compared to control cells (set at 100). (C) Sparsely seeded cells were pretreated with TGF-β (400 pM) for 16 h, followed by addition of HGF (220 pM) without removal of TGF-β, and incubations were continued for the indicated times (4 to 36 h). Untreated control cells are included (lanes 1 and 14). In wash , cells treated with TGF-β for 16 h were washed four times with growth medium, followed by addition of fresh medium. The cells were lysed, followed by Cdk6 and Cdk2 immunoblotting and determination of Cdk2-associated kinase activity towards histone H1. Relative kinase activities after subtraction of the kinase activity given by nonimmune serum are shown compared to cells treated with TGF-β for 16 h (set at 100) (lane 1). 5-BrdUrd incorporation was analyzed for the last hour of each incubation. The results are expressed as percent 5-BrdUrd-positive nuclei of Hoechst 33258-stained nuclei.