Rap1 activation in neutrophils. Neutrophils were stimulated with either 1 μmol/L fMLP, 1 μmol/L PAF, or 0.1 nmol/L GM-CSF for indicated time points. Cells were lysed and Rap1 GTP was isolated using His-Ral-GDS RBD. Rap1 was detected by Western blot analysis using a monoclonal antibody against Rap1. The amount of Rap1 GTP isolated after 10 seconds of fMLP stimulation and unstimulated neutrophils was compared with the amount of total Rap1 (Rap1GDP and GTP) present in various amounts (%) of total lysate. The experiments were repeated at least three times with similar results.