FIGURE 8.

WW2-EGFP and EGFP-Pro function as dominant negative mutants in the Gas7-induced membrane protrusion. A, extracts from COS-7 cells co-transfected with HA/N-WASP, and either EGFP (negative control) or WW2/EGFP was analyzed by co-IP to assay the interaction between N-WASP and WW2-EGFP. A specific interaction between N-WASP and WW2-EGFP was found. B, extracts from COS-7 cells co-transfected with Gas7/Myc and either EGFP (negative control) or Pro/EGFP were analyzed by co-IP to assay the interaction between Gas7 and Pro-EGFP. A strong interaction between Gas7 and Pro-EGFP was detected. C, immunostaining of Neuro-2a cells co-transfected with Gas7/Myc and either Gas7 dominant negative (WW2/EGFP) or N-WASP dominant negative (EGFP/Pro) mutants to examine competition performance in Gas7-induced membrane protrusion. Unlike control (Gas7/EGFP) cells, Gas7/WW2-EGFP and Gas7/EGFP-Pro co-transfected Neuro-2a cells did not show any membrane protrusions. Scale bar, 10 μm. D, fractions of cells in C with membrane protrusions are shown. Data are presented as the mean ± S.E. of three independent determinations, each counting at least 50 cells. Statistically significant decreases of the fraction of cells that extended processes compared with the control group (EGFP/Gas7 co-transfection) are indicated (paired Student's t test; **, p < 0.01).