Fig. 6.

Effects of the N-alkoxy I3C derivatives on CDK2 specific kinase activity. MCF-7 cells were treated with 200 μM I3C, 10 μM N-methoxy I3C, 5 μM N-ethoxy I3C, 1 μM N-propoxy I3C, 0.5 μM N-butoxy I3C, 100 μM tryptophol, or with the DMSO vehicle control for 48 hr, and CDK2 was immunoprecipitated from total cell extracts and immunoprecipated with anti-CDK2 antibodies, one control sample did not receive any CDK2 antibodies (No IP). The immunoprecipitated CDK2 protein was assayed for in vitro kinase activity using 0.21 μg Rb carboxy-terminal domain protein as a substrate in each reaction mixture. The kinase reactions were electrophoretically fractionated and the level of [³²P]Rb was visualized by autoradiography. The efficiency of immunoprecipitation for each sample was confirmed by western blot analysis using antibodies specific to CDK2.