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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.
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