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. 2006 Jun 16;7(7):704–709. doi: 10.1038/sj.embor.7400737

Figure 1.

Figure 1

Sema4D suppresses R-Ras activity, dephosphorylates Akt and GSK-3β and phosphorylates CRMP2 in cultured hippocampal neurons. Neurons dissociated from rat embryos at embryonic day 18.5 were stimulated at 3 days in vitro (d.i.v.) with Sema4D for 1.5 h. (A) Activity of R-Ras with or without Sema4D stimulation. GTP-bound R-Ras isolated with GST–RBD was detected with an anti-R-Ras antibody. Relative activity of R-Ras was determined by the amount of R-Ras bound to GST–RBD normalized to the amount of R-Ras in cell lysates analysed by National Institutes of Health Image software. (B–D) Analysis of phosphorylated (p)-Akt, p-GSK-3β and p-CRMP2. Cell lysates were analysed by immunoblot analysis with the phospho-specific antibodies against Akt (Ser 473; B), GSK-3β (Ser 9; C) and CRMP2 (Thr 514; D). Results are the means±s.e.m. of three independent experiments. CRMP2, collapsin response mediator protein 2; GSK-3β, glycogen synthase kinase-3β; GST–RBD, glutathione S-transferase-fused Ras-binding domain; Sema4D, semaphorin 4D.