Fig. 5. Localization of active Rho GTPases in vivo at E11-E13. (A) Rhombic lips from E11 were lyzed and incubated with GST-RBD-Rhotekin protein. GTP-bound RhoA (active) was detected by western blot using an anti-RhoA antibody and compared with total RhoA contained in cell lysates before the incubation with GST-RBD-Rhotekin. (B) Section through the caudal hindbrain at E11 after incubation with RBD-Rhotekin fused to the GST and then treatment with an anti-GST antibody. A strong RhoA/B/C GTPase activity was observed in the ventricular zone (vz) and the dorsal border (arrow) of the rhombic lip. The marginal (mz) and submarginal (smz) zones also showed GST-RBD-Rhotekin labeling. No staining could be observed in the subventricular zone (svz). (C) Higher magnification of image in B, showing scattered GST-RBD-Rhotekin-positive cells (arrowheads) leaving the ventricular zone (large arrow) toward the early migrating stream (short arrows). (D) At E13, migrating LRN located in the marginal stream showed strong GST-RBD-Rhotekin binding (arrowhead). ION (asterisk) reaching the vicinity of the floor plate showed faint Rho GTPase activity. (E,F) Control experiments (CTRL) were performed with a non-relevant GST-tagged protein (M-Cadherin) at E11 (E) and E13 (F). No staining could be observed. (G) The expression patterns of RhoA/B GTPases and active RhoA/B proteins at E11 and E13. Scale bars: B,E, 200 µm; C, 500 µm; D,F, 260 µm.