Figure 2.

Characterization of MEL cell transfectants. (A) MEL cell transfectant clones expressing the indicated CDKI under control of the tetracycline-inducible promoter in pUHD 10-3 were cultured either in the absence (−) or in the presence (+) of 1 μg/ml doxycycline (dox) for 36 h. p16 MEL cell transfectants were first cultured in HMBA for 84 h, at which point endogenous p16 is present at very low levels (Fig. 1). Total cellular-protein extracts were prepared, and the levels of the CDKIs were determined by immunoblotting. The parental MEL cells (clone B1) containing only the rtTA regulator were cultured in the absence (0) or presence (120) of 5 mM HMBA for 120 h to indicate the levels of endogenous p16 in undifferentiated cells or that of p15, p21, and p27 present in fully differentiated MEL cells. For further details see Materials and Methods. (B) The indicated MEL cell transfectants were cultured in the presence (+) or absence (−) of doxycycline for 36 h as described in Materials and Methods. MEL and MEL rtTA cells were cultured in the presence (+) or absence (−) of roscovitine for 24 h as described in Materials and Methods. Extracts were prepared and immunoprecipitated-Kinase assays were performed as described in Materials and Methods. (C) p21 MEL cell transfectants (clone p21.12 and p21.35) were cultured in the presence (+) or absence (−) of doxycycline for 36 h. Total cellular protein extracts were prepared and immunoprecipitated (IP) with antibodies specific for the indicated CDKI as described in Materials and Methods. The immunoprecipitates were subjected to SDS/PAGE and immunoblotted for the indicated CDK.