Analysis of Erk activation in immature and differentiated hematopoietic cells. Serum-starved cells were incubated 30 minutes with 20 μM LY294002 (lane 3) or 50 nM wortmannin (lane 4), as indicated (+) prior to 5 minutes of stimulation with 50 ng/mL SF (lanes 2 to 4). Cell lysates (20 μg per lane) were subjected to SDS-PAGE and then immunoblotted with antiphospho-specific Erk (left panels) and then stripped and reprobed with anti-Erk (right panels), as indicated. Cells analyzed were as follows: (A) ES-HPCs; (B) bone marrow–derived HPCs (BM-HPC); (C) mast cells differentiated from bone marrow cells (BM-mast); (D) mast cells obtained from differentiated HPCs (ES-HPC-mast); (E) myeloid-committed cells obtained from differentiated HPCs (ES-HPC-myeloid); (F) mast cells generated from Lhx2-transduced ES cells (ES-Lhx2-mast); and (G) mast cells generated from vector-transduced ES cells (ES-vec-mast).