G-CSF– but not IL-3–induced Erk1/2 activation in Gfi1-null bone marrow cells is impaired, correlating with defective Ras activation. (A) Immunoblot analysis of IL-3–induced signaling responses in bone marrow MNCs from Gfi1^+/+, Gfi1^+/−, and Gfi1^−/− mice. Freshly obtained cells were incubated with for 10 minutes in either medium only or with IL-3 (10 ng/mL). The results reflect reprobing of a single membrane. The results are representative of 7 experiments. (B) Phospho-Erk1/2 activation by IL-3 or G-CSF in Gfi1^+/+ and Gfi1^−/− bone marrow MNCs evaluated by immunoblotting with specific antibodies. Cells were incubated for 10 minutes in medium only or with IL-3 (10 ng/mL) or G-CSF (25 ng/mL). The results are representative of 5 experiments. (C) Active Ras was pulled down from bone marrow cell lysates with agarose-bound Raf-1 protein (residues 1-149 corresponding to the binding domain for Ras-GTP) and immunoblotted with Ras-specific antibody. Bone marrow MNCs from 2 Gfi1^+/+ and 2 Gfi1^−/− mice were incubated in medium only or with G-CSF (25 ng/mL) for 10 minutes; the cell lysates were either immunoblotted directly or after Ras-GTP pull down. The membrane was reprobed with specific antibodies to phospho-Erk1/2 or total Erk. The results are representative of 3 experiments performed.