Fig. 9. Inhibition of ERK activation lead to site-specific Rb hypophosphorylation. Rb expression was induced in the 10D5 subclone by a 24-h incubation in serum-containing medium with doxycycline followed by serum-starvation for 6 h. The cells were then placed in serum-free medium containing 100 ng/ml SCF and 1 mCi/ml [32P]Pi in the presence of DMSO or 80 µM PD 98059. The incubation was continued for 12 h, at which time Rb was immunoprecipitated, electrophoretically resolved, and analyzed by autoradiography and immunoblotting using antibodies directed against total Rb and phospho-specific isoforms. After probing for total Rb protein, the blot was stripped and stained with a pool of three antibodies directed against phospho-peptides containing pSer807/811, pSer780, and pSer795. The blot was then stripped and stained sequentially with each antibody individually (in the above order). The staining with the pSer807/811 antibody is not shown because it was completely negative. Rb phosphorylated on Ser795, present in the slower mobility band, is selectively lost after treatment with PD 98059.