Activation of p21rac by diverse stimuli in human neutrophils. (A) Neutrophils were isolated as described and stimulated with either PAF (1 μmol/L), GM-CSF (10⁻¹⁰ mol/L), or TNF- (100 U/mL) for the indicated time. Cells were lysed and active p21rac was isolated using GST-PAKcrib as described in Materials and Methods. p21rac was detected by Western blot analysis. (B) Neutrophils were stimulated with either GM-CSF (10⁻¹⁰ mol/L) for the indicated time or with fMLP (1 μmol/L) for 10 seconds. Cells were lysed and active p21ras was isolated using GST-Raf1-RBD. p21ras was detected by Western blot analysis. (C) Eosinophils were stimulated with either fMLP (1 μmol/L) or PAF (1 μmol/L) for 10 seconds. Cells were lysed and active p21rac was detected as above.