Figure 1.

Oncogenic stimulation invokes Cdk6 to participate in the cell cycle start. NRK cells and the mutants 39–1 and 23 were arrested in G₁ by serum depletion and stimulated to resume cell cycling by the addition of FCS alone, FCS containing TGF-β (T), or FCS containing EGF plus TGF-β (E+T). The cells then were microinjected with antibodies and those that had entered S phase were identified by BrdUrd incorporation followed by immunostaining with the anti-BrdUrd mouse antibody ZBU30 (Seikagaku) and, subsequently, with a Texas Red-conjugated anti-mouse IgG antibody (34). The antibodies injected were control IgG, dilute αCdk4 (0.1 mg protein/ml), αCdk4 (1 mg protein/ml), epitope peptide-neutralized αCdk4, αCdk6 (1 mg protein/ml), an equal mixture of αCdk4 and αCdk6 (0.5 mg protein/ml each), and an equal mixture of epitope peptide-neutralized αCdk4 and αCdk6. αCdk4 and αCdk6 were similar in potency as judged by the ability to immunoprecipitate Cdk4 or Cdk6 molecule (15). Microinjected cells were identified by immunostaining with an FITC-conjugated anti-rabbit IgG antibody. The values shown were averaged, and the SDs (error bars) were calculated from the results in three to five independent experiments.