An EPOR-PY343 STAT5 axis mediates GAB2 but not AKT activation. (A) Kit^posCD71^highTer119^neg erythroblasts were prepared from wt-EPOR, EPOR-H, and EPOR-HM bone marrow cell expansion cultures. Cells were then cultured for 5.5 hours in IMDM containing transferrin (50 μg/mL), insulin (15 ng/mL), and BSA (0.5%). EPO (2 U/mL) was then added and at 0, 35, and 75 minutes lysates were prepared and levels of PY452 GAB2 and total GAB2 were determined by Western blotting. Outcomes were analyzed quantitatively by scanning densitometry (bottom panel). (B) Coupling to AKT is deficient for not only EPOR-HM but also EPOR-H alleles. Bone marrow–derived Kit^posCD71^high erythroblasts were prepared and purified (as described in panel A) from EPOR-HM, EPOR-H, and wt-EPOR mice. Following withdrawal of hematopoietic cytokines (for 5.5 hours), cells were exposed to EPO (2.5 U/mL) and at the indicated intervals lysates were prepared. Levels of phospho-S473-AKT then were determined by Western blotting (and normalized for AKT levels/loading). Note the limited activation of AKT in not only EPOR-HM but also EPOR-H erythroblasts. Vertical lines indicate reassembled segments from a single, uniform en-hanced chemiluminescence (ECL) exposure. (C) Bone marrow–derived Kit^posCD71^highTer119^neg erythroblasts expressing an EPOR-H allele are efficiently protected by EPO against apoptosis, while EPOR-HM erythroblasts are not; Kit^posCD71^high wt-EPOR, EPOR-H, and EPOR-HM erythroblasts were expanded and purified. Cells were then treated with LY274002 to inhibit PI3K,⁵⁶ and were cultured in the presence of EPO at 0.2 U/mL. At 18 hours of culture, frequencies of apoptotic cells were determined by annexin-V staining and flow cytometry. Representative outcomes for LY274002 dosing at 15 and 50 μM are shown.