Fig. 5. RhoA activation is critical for mediating the effect of Nogo-66 on OECs. (A,B) Y-27632 treatment significantly attenuates the enhancement of Nogo-66 on adhesion of OECs. OECs (a,c,e,g,i) and Y-27632-treated OECs (b,d,f,h,j) were plated on dishes coated with laminin (a,b), GST (c,d), GST–Nogo-66 (e,f), His (g,h) or His–Nogo-66 (i,j). After incubation for 30 minutes with gentle shaking, adherent cells were photographed and counted. The values were normalized with respect to the values obtained for laminin. (C) Y-27632 treatment significantly attenuates the inhibition of Nogo-66 on migration of OECs. OECs were pre-treated with or without Y-27632 and applied to Boyden chamber assays. The trans-well membranes were coated with laminin, GST, GST–Nogo-66, His or His–Nogo-66 (100 µg/ml each). Values were reported as mean ± s.d./visual field. ^*P<0.05, ^**P<0.01 versus laminin or vehicle. (D) RhoA activation in OECs was assessed with pull-down assays with the Rho-binding domain of rhotekin. The amount of GTP-bound RhoA was normalized to the amount of total RhoA. Results are means ± s.d. from three independent experiments. ^*P<0.05 versus GST or His. (E) Nogo-66 promotes the formation of focal adhesions and inhibits membrane protrusion of OECs through RhoA activation. OECs and Y-27632-treated OECs were plated for 60 minutes on coverslips coated with GST (a-d), GST–Nogo-66 (e-l), His (m-p) or His–Nogo-66 (q-x). Cultures were then fixed, permeabilized, and stained with anti-paxillin to visualize focal adhesions (green) and rhodamine-conjugated phalloidin to visualize filamentous actin (red). Arrows (in c,k,o,w) indicate the thick bundles of actin in the membrane protrusions of OECs. Arrowheads (in h,t) indicate the stress fibers terminating at the adhesion plaques. Bar, 10 µm. (F) Quantification of the paxillin punctae. The number of punctae/cell were counted for 40 cells selected randomly in each group. The number of punctae was normalized by comparing with GST or His, and the percentage of punctae in control and treated cells was compared. ^**P<0.01.