Characterization of p18 and p27 MEL cell transfectants.

(A) MEL cell transfectant clones expressing either p18 or p27 under control of the tetracycline inducible promoter in pUHD 10-3 were cultured either in the absence (−) or presence (+) of 1 μg/mL Dox for 36 hours. Total cellular protein extracts were prepared, and the levels of p18 (left panel) and p27 (right panel) were determined by immunoblotting. The parental MEL cells (clone B1) containing only the rtTA regulator were cultured in the presence of 5 mmol/L HMBA for 120 hours to indicate the levels of endogenous p18 and p27 present in fully differentiated MEL cells. For further details see “Materials and methods.” (B) The effect of exogenous p18 and p27 on CDK activities in undifferentiated and differentiated MEL cell transfectants. Extracts prepared from the indicated transfectant clones cultured in the presence of 5 mmol/L HMBA and either in the absence (−) or presence (+) of Dox were prepared at the indicated times and assayed for the indicated CDK activity by IP-kinase assays as described in “Materials and methods.”