EpoR-HM, EpoR-H, and wt-EpoR modulation of p70S6-kinase, Akt, and p60-Src. (A) p70S6-kinase activation via the wt-EpoR, but not EpoR-H or EpoR-HM. Erythroblasts from wt-EpoR, EpoR-HM, and EpoR-H mice were expanded, washed, cultured for 6 hours in the absence of cytokines, and then stimulated for the indicated intervals with Epo (2.5 U/mL). Lysates then were prepared and levels of phosphorylated and total p70S6-kinase were assayed by Western blotting. (B) Deficient Akt activation via EpoR-HM and EpoR-H alleles. Erythroblasts were prepared as described for panel A, exposed to Epo, and analyzed for Akt activation. Note the multifold deficit activation of Akt via EpoR-HM and Epo-H alleles. (Ci-ii) Deficient PY-416 p60-Src expression via EpoR-HM. In the wt-EpoR, EpoR-HM, and EpoR-H mouse-derived cells and samples (B), levels of phospho-p60-Src (and p60-Src) were assayed by Western blotting (and digital densitometry imaging). (Ciii) Differential PP2 inhibition of EpoR-HM, EpoR-H, and wt-EpoR erythroblast expansion. During in vitro expansions, wt-EpoR, EpoR-H, and EpoR-HM erythroblasts were exposed to 15 μM PP2. Effects on erythroblast formation were assessed by direct cumulative cell counts at day 3 of expansion and are normalized to numbers for parallel DMSO-exposed control cultures. PP3 also was tested but was without significant effects (data not shown). Plotted values are mean values plus or minus SE; n = 3 wt EpoR, EpoR-HM, and EpoR-H mice per group.