Figure 9.

NOMA-GAP negatively regulates Cdc42 and PAK downstream of NGF. (A and B) PC12 cells were transfected with control (C) or rat NOMA-GAP (N) siRNA in the absence (A) or presence (B) of expression constructs for wt and Y1169A huNOMA-GAP as described in Fig. 8 G. Lysates were immunoblotted as indicated. Vertical lines denote nonconsecutive lanes from the same gel and Western blot. (C) GST-PAK CRIB pull-downs were performed on lysates of PC12 cells transfected 24 h earlier with control or rat NOMA-GAP siRNA and stimulated with NGF. Pull-downs and lysates were immunoblotted as indicated. (D and E) PC12 cells were transiently transfected with dominant-active (V12) Cdc42, wt NOMA-GAP, and a GFP transfection marker as indicated and stimulated with NGF 24 h after transfection. (D) Samples were stained 72 h after transfection for polymerized actin (red) and GFP (green). Bar, 20 μm. (E) Quantification of the proportion of cells forming large lamellipodia (>30 μm diameter) in duplicate samples. (F–H) PC12 cells were transfected with control siRNA or NOMA-GAP siRNA in the presence of increasing levels of an expression construct for Myc-tagged dominant-negative (N17) Cdc42 and a GFP transfection marker. Cells were stimulated with NGF 4 h after transfection and were then either lysed 48 h after transfection and analyzed for expression of Myc-tagged N17 Cdc42 (F) or stained 72 h after transfection for polymerized actin (red) and GFP (green) (H). Bar, 20 μm. Asterisk marks a nonspecific band. (G) Quantification of the proportion of NGF-stimulated control or NOMA-GAP siRNA-transfected cells bearing neurites (>30 μm) in the presence of increasing levels of N17Cdc42 or N17Rac. The normalized average of two independent experiments is shown.