STAT and Erk activation. (A) Bone marrow cells were stimulated with G-CSF (100 ng/mL) for the indicated time (in minutes) and EMSA assays performed with oligonucleotide probes specific for STAT-3 (left panel) and STAT-5 (right panel). The identity of the complexes was confirmed by supershifting with appropriate antibodies (data not shown). (B) c-Kit⁺ Lin^- cells were isolated from the bone marrow and cultured for 5 days in the presence of kit ligand and G-CSF to generate a cell population enriched in granulocytic precursors. After starvation for 4 hours in cytokine-free media, the cells were stimulated with G-CSF (100 ng/mL) for the indicated time (in minutes) and Western blot assays performed to measure phosphorylated Erk (P-Erk) or total Erk (Erk) protein. Molecular weight markers (in kDa) are shown at the left of each blot. Data are representative of 3 independent experiments.