Comparison of exogenous and endogenous kinase activities in undifferentiated and differentiated MEL cell CDK transfectants.

The indicated MEL cell transfectants were cultured in the absence (0) or presence (120) of HMBA for 120 hours and then further cultured in the absence (−) or presence (+) of Dox for 36 hours. Cell extracts were prepared and immunoprecipitated with antibodies specific for the endogenous murine CDKs (K2, K4, and K6) or the exogenous human HA-tagged CDKs (HA or HuK2). In doubly transfected cells (panels B and C) assays of exogenous kinase activities were performed by first immunoprecipitating with an antibody specific for the exogenous human CDK2 (HuK2), and after checking that the exogenous CDK2 was completely removed, the supernatant was immunoprecipitated with an anti-HA antibody specific for the exogenous HA-tagged (B) CDK4R24C or (C) CDK6R31C. The immunoprecipitates were incubated with γ-³²P–ATP (γ-phosphorous 32–adenosine 5′-triphosphate) in the presence of either H1 or the GST-Rb C-terminal fragment, and the reactions were resolved by SDS-PAGE. For further details see “Materials and methods.”