Figure 4. Role of Rac GTPase in myelin ovoid formation in vivo. A, Rac and Cdc42 activity assay. Levels of active Rac1 (Rac1-GTP) and Cdc42 (Cdc42-GTP) were measured using a pull-down assay. B, Immunofluorescent localization of Rac1 and Cdc42 in the rat sciatic nerves following nerve injury. Arrows indicate SLIs, whereas asterisks indicate Ranvier nodes. C, New F-actin formation assay. Explants were treated with jasplakinolide for 3 h in vitro, and then the explants were further incubated for 6 h in the presence or absence of NSC23766. D, Myelin ovoid index showing the effect of NSC23766 and Y27632 (a Rho kinase inhibitor). E, Explants cultured for the indicated time in the presence of PKI166 were assayed for Rac activity. F, G, Transfection of a dominant-negative Rac1-GFP (RacDN) or siRNA for Rac1 (G) inhibited formation of myelin ovoids. GFP indicates a GFP-vector control. G, Quantitative result showing the relative number of myelin ovoids to GFP or scrambled RNA (SCR) transfected controls. Western blot shows the effect of Rac1-RNAi on Rac1 expression in the transfected sciatic nerves. H, After sciatic nerve axotomy, the lesioned nerve was treated with CD or vehicle, and nerves were processed for immunofluorescent staining against E-cadherin. I, Myelin ovoid index from lesioned nerve in vivo. Scale bar, 50 μm.