"FN-induced EGFR activation is required for G1 cell cycle entry. (A and B) Adherent CV1 cells were serum starved for more than 48 h (Go), placed in suspension (S), left untreated or treated with 1 μM AG1478 (AG), and plated on FN in the absence (FN) or presence (EGF) of 40 ng of EGF/ml for 12 or 18 h. The time points selected were determined to be optimal for the events monitored in time course studies (data not shown). (A) The levels of <t>cyclin</t> <t>D1</t> (cycD1), cyclin E (cycE), p27, p21, and Rb phosphorylation at Ser807/811 (P-Rb) at 12 h and the levels of cyclin A at 18 h were monitored by immunoblotting cell lysates with their respective antibodies. Total levels of Akt were monitored by immunoblotting as a control for total protein levels in the extracts. (B) cdk2 activity was measured by using in vitro kinase assays with histone H1 as a substrate and was quantified (1.74- ± 0.03-fold induction in the presence of FN only; 2.69- ± 0.01-fold in the presence of FN and EGF; n = 3). Preimmune (PI) and no-substrate (NS) controls were included. Total levels of kinase immunoprecipitated in the assays were monitored by immunoblotting with anti-cdk2 antibodies (cdk2). (C and D) CV1 cells were prepared as described above except that cells in suspension were pretreated with either 10 μM U0126 (U0), 100 nM wortmannin (WT), or 0.5 μM PD168393 (PD) 15 min prior to plating on FN. Results obtained after plating for 12 h are shown. (C) cdk4 kinase activity was monitored by an in vitro kinase assay by measuring phosphate incorporation into GST-Rb 12 h after plating on FN and was quantified (1.4- ± 0.2-fold in the presence or absence of EGF; n = 3). Preimmune and no-substrate controls were included. Total levels of kinase immunoprecipitated in the assays were monitored by immunoblotting with anti-cdk4 antibodies (cdk4). (D) Levels of cyclin D1, p27, and Rb phosphorylation at Ser807/811 were monitored by immunoblotting cell lysates with their respective antibodies. Total levels of Akt were monitored by immunoblotting as a control for total protein levels in the extracts. "