Figure 5.

NogoΔ20-induced Rho activation depends on internalization. (A–C) Rho activation levels were examined in PC12 cells that were either untreated (control; A), treated with 300 nM NogoΔ20 for 30 min at 37°C in absence of mutant PincherG68E (B), or treated with 300 nm Nogo Δ20 treated for 30 min at 37°C in the presence of mutant PincherG68E (C). Active GTP-bound Rho was detected by incubation with GST-tagged Rhotekin-RBD and immunostaining. Bar, 20 µm. (D) Densitometric quantification of staining from three independent experiments. Data are normalized to the mean of the untreated group ± SEM (error bars); asterisks marks highly significant differences between untreated, NogoΔ20-treated, or NogoΔ20- and dn PincherG68E-treated cells (three experiments; 30–50 cells per experiment; ***, P < 0.001; Student’s t test). (E) PC12 cells were either left untreated or transfected with dn PincherG68E construct. All cells were then incubated with 300 nM NogoΔ20 for 30 min at 37°C. Extracted proteins were precipitated with Rhotekin-RBD beads. Precipitates were immunoblotted for RhoA (top). Total RhoA levels were determined from whole cell lysates as shown in the bottom panel.