Fig. 11. Rap-1 activation is impaired in JAM-A^–/– neutrophils. A pull-down assay was performed to isolate active GTP-bound Rap-1. Freshly isolated mouse neutrophils were obtained from JAM-A^+/+ and JAM-A^–/– mice. 5x10⁶ cells for each condition were left untreated or stimulated with the chemotactic peptide WKYMVm for 5 minutes at 37°C. Pull down was performed on cell lysates using the RalGDS-RBD probe (A) or the GST-negative control (C). Western blot for Rap-1 revealed impaired Rap-1 activation in both unstimulated and stimulated JAM-A^–/– compared with JAM-A^+/+ neutrophils. (B) Densitometric analysis of active GTP-bound Rap-1 normalized for the total amount of protein. Results in B are means ± s.e.m. of four separate experiments; A and C show a typical experiment.