Figure 2.
Effect of phosphorylation-resistant pRB mutants on cytokine-dependent 6–1 cell growth. (A) Induction of pRBΔp34HA in cycling (Top) or growth-arrested (Middle) transfectants, Δp34HA-9 and Δp34HA-14, was examined by anti-pRB immunoblotting. Positions of the hypophosphorylated pRB (pRB), the phosphorylation-resistant pRB mutant (Δp34-HA), and the hyperphosphorylated pRB of endogenous origin (ppRB) were indicated. The growth-arrested cells were then restimulated with various doses of IL-2, and cell cycle entry and progression triggered by IL-2 were examined by [3H]thymidine incorporation (Bottom). (B) The pRBΔp34HA-transfectants were cultured in medium containing IL-2 or IL-3 in the presence of Tc or IPTG, and cell numbers were counted. (C) Induction of pRBΔS/T-P, which has an HA epitope at the N terminus, in asynchronously growing transfectant cells in the presence of Tc or IPTG was examined by immunoblotting with anti-pRB or anti-HA. (D) The pRBΔS/T-P-transfectants were cultured in medium containing IL-2 or IL-3 in the presence of Tc or IPTG, and cell numbers were counted.