Fig. 2.

Modulation of CDK4 by ST is pRb dependent. (A) 76NE6 and (C) 76NE7 cells were infected with adenoviral vectors containing 1000 MOI of CDK4 or LUC for 48h and were then treated with a range (0–4nM) of ST concentrations for an additional 24h. The cells were harvested and protein lysates were collected and subjected to western blot analysis with CDK4. CDK4 was immunoprecipitated from the lysates and tested for kinase activity against GST-Rb (B and D). Blots are shown in the bottom panel with densitometric values graphed above. The specificity of the CDK4 kinase assay was assessed using a CDK4/6 inhibitor, PD0332991, in the lanes marked with an asterisk (panel D*). The background GST-Rb kinase band in the PD0332991 lanes was subtracted from the other lanes to generate the densitometric values presented in panel D. The cell lysate from each cell line was also subjected to IP with CDK4 followed by western blot to show the amount of total protein subjected to the kinase assay (panel B and D, bottom panel). MCF-7 control and shRNA-CDK4 cells were treated with increasing concentrations of ST for 24h, cells were harvested and subjected to (E) western blot and (F) flow cytometry. To show the degree of downregulation rendered by shRNA-CDK4, an untreated aliquot of MCF-7 control cells was loaded on the same gel as the ST-treated MCF-7 CDK4-shRNA gel.