17-AAG-induced G1 cell cycle block is due to downregulation of specific Hsp90 clients. (A) Detection of Cdk4 protein (Western blotting) and Cyclin D1 gene (RT-PCR), both critically implicated in the G1 to S phase transition, over a 24-hours treatment period of human bladder cancer cells with 17-AAG. The mRNA expression levels of the internal control gene GAPDH were also quantified by an RT-PCR approach. (B) Western blotting of the cell cycle regulatory proteins pRb and E2F1, after exposure of RT4, RT112 and T24 cells to different 17-AAG doses. Immunoblotting and RT-PCR experiments were repeated three times.