Figure 6.

In primary erythroid progenitors, conditional deletion of Spry1 derepresses Jak2. (A) Erythroid progenitors from the bone marrow of control (Spry1^flox/flox) or Spry1-deleted mice were expanded via short-term culture in SP34ex. Lin⁺ cells were then depleted (including a second round of Ter119⁺ cell depletion). Kit⁺CD71^highTer119⁻ stage E1 cells were then isolated and cultured for 5.5 hours in the absence of hepatocyte growth factors. Cells then were exposed to EPO (1 U/mL for 0, 8, or 24 minutes), and lysates were directly prepared. Levels of PY-1007, 1008; Jak2 plus Jak2; P-Ser473-Akt plus Akt; and P-T202, P-Y204 Erk1,2 plus Erk1,2 then were determined by ECL Western blotting. Right panel: fold modulation of Jak2 PY-1007, 1008 phosphorylation by EPO in control versus Spry1-deficient cells is quantitatively analyzed (at 8 plus 24 minutes of EPO exposure). (B-C) Proposed routes for Spry1 regulation of erythropoiesis. (B) A model is outlined via which Spry1 acts to inhibit Jak2 as well as Erk1,2. (C) Routes for Spry1 regulation of proerythroblast expansion are also outlined, including heightened E1 and E2 cell expansion resulting from Spry1 disruption. A potential limiting of late-stage erythroblast (and red cell progeny) formation by Spry1 is also shown.