Ability of mutated EpoR/gp130 chimeras to mediate survival and proliferation. (A) Schematic representation of the mutations introduced into EpoR/gp130 (Eg). Tyrosine (Y) to phenylalanine (F) substitutions are printed in light face. The ability of the gp130 mutants to recruit downstream effector molecules is outlined. (B) INA-6 strains expressing the EpoR/gp130 chimeras as indicated were deprived of IL-6 for 12 hours before stimulation with Epo (7 U/mL) for another 8 hours. Thereafter, cell lysates were applied to immunoblot analysis by using antibodies against Stat3 or tyrosinephosphorylated Stat3. Cytokine-starved wild-type Eg strain was taken as a reference. (C-D) INA-6 and XG-1 strains were cultured without or with Epo for 24 hours (INA-6) and 72 hours (XG-1) in the absence of IL-6 and subsequently subjected to an apoptosis assay. The graph represents the percentage of annexin V–positive cells (mean values ± SD of 3 experiments) in the Epo-treated cultures as compared with those cultured without cytokine. (E) XG-1 strains were cultured without or with Epo for 72 hours in the absence of IL-6. Then, a flow cytometric cell cycle analysis was performed. The ratio of cells in G1 to those in S + G2 phases is shown (mean values ± SD of 3 experiments). n.d. indicates not determined.