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. 2009 May 15;10(6):614–621. doi: 10.1038/embor.2009.63

Figure 1.

Figure 1

Plexin-B1 shows GTPase activating protein activity for M-Ras. (A) COS-7 cells expressing Myc-Plexin-B1 (WT or RA mutant), green fluorescent protein (GFP)-Rnd1 and indicated HA-tagged Ras GTPases were stimulated with Sema4D. The cell lysates were incubated with glutathione S-transferase-fused Ras-binding domain (GST-RBD) and bound Ras GTPases; total cell lysates were analysed by immunoblotting. Relative Ras activities were determined by the amounts of Ras bound to GST-RBD normalized to the amount of Ras in cell lysates analysed by National Institutes of Health Image software. (B) Recombinant Myc-tagged cytoplasmic domain of Plexin-B1 (WT, RA or GGA mutant) and GTPγS-loaded Rnd1 were incubated with M-Ras preloaded with GDP or GTPγS, and then immunoprecipitated (IP) with an antibody against Myc. Bound and total proteins were analysed by immunoblotting with the indicated antibodies. (C) Lysates from transfected COS-7 cells expressing the indicated proteins were immunoprecipitated with an antibody against Myc, and bound and total proteins were analysed by immunoblotting with the indicated antibodies. (D) COS-7 cells, transfected with the indicated plasmids, were stimulated with Sema4D. The cell lysates were incubtated with GST-RBD and bound M-Ras; total cell lysates were analysed by immunoblotting. Results are the means±s.e.m. of three independent experiments. GAP, GTPase activating protein; HA, haemagglutinin; WT, wild type.