Epo receptor allele activation of JNKs and ERKs. (A) Efficient JNK activation via the wt-EpoR, but not via EpoR-H or Epo-HM alleles. Erythroblasts expanded from wt-EpoR, EpoR-HM, and EpoR-H bone marrow preparations were washed, cultured for 6 hours in the absence of cytokines, and stimulated with Epo (2.5 U/mL) for the indicated intervals. Levels of phospho-JNKs (and total JNKs) were assayed by Western blotting and digital densitometry imaging. The top panel (Ai) illustrates results for the wt-EpoR and includes coanalyzed positive controls (no. 9253; Cell Signaling). In the bottom panel (Aii), note the nominal activation of JNKs via EpoR-HM and Epo-H alleles. (B) ERK hyperactivation via EpoR-HM. (Bi) Erythroblasts expanded from wt-EpoR, EpoR-HM, and EpoR-H bone marrow preparations were washed, cultured for 6 hours in the absence of cytokines, and stimulated with Epo (2.5 U/mL) for the indicated intervals. Levels of phospho-ERK1,2 (and total ERK1,2) were assayed by Western blotting and digital densitometry imaging. (Bii) Parallel analyses of EpoR allele activation of ERKs were performed using expanded, Ter119-depleted wt-EpoR, EpoR-HM, and EpoR-H CD71^high erythroblasts (and Epo exposure was extended to 60 minutes). For comparison, levels of Epo-stimulated phosphorylated p38-MAPK (PT180 and PY182) (and total p38-MAPK) also were assayed (Figure S3).