Immunoblot analysis of murine BM culture after GM-CSF or IL-3 stimulation. (A) The BM cells were incubated in the presence of GM-CSF or IL-3 for 3, 5, 7, and 10 days. Cell lysates (20 μg/lane) were analyzed by immunoblotting with anti-murine FAK rabbit antibody JF1 (upper panel) and reprobed with anti-Mac-1 Ab (lower panel). Lanes 1 and 2, unstimulated BM; lanes 3 through 10, stimulated by GM-CSF; lanes 11 through 18, stimulated by IL-3. Lanes 1, 3, 5, 7, 9, 11, 13, 15, and 17, adherent cells; lanes 2, 4, 6, 8, 10, 12, 14, 16, and 18, nonadherent cells. Arrowhead indicates FAK protein (125 kD). (B) Immunoblot analysis of murine BM before and after cytokine switching. The BM cells were incubated with GM-CSF or IL-3 for 5 days, before switching to the other growth factor for another 5 days. Cell lysates (20 μg/lane) were immunoblotted with JF1 anti-FAK Ab (upper panel) and reprobed with anti-Mac-1 Ab (lower panel). Lanes 1 and 2, unstimulated BM; lanes 3 and 4, day 5 cell lysates after stimulation with IL-3 only; lanes 5 and 6, cytokine switching from IL-3 to GM-CSF; lanes 7 and 8, day 5 cell lysates after stimulation with GM-CSF only; lanes 9 and 10, cytokine switching from GM-CSF to IL-3. Lanes 1, 3, 5, 7, and 9, adherent cells; lanes 2, 4, 6, 8, and 10, nonadherent cells. Arrowhead indicates FAK protein (125 kD).