Fig. 6. Antagonistic effects of N-WASP and dynamin 2 on SNX9-induced tubulation. (A,B) When high levels of GFP-tagged N-WASP or mRFP-tagged dynamin 2 were coexpressed with GFP- or mRFP-tagged SNX9 in COS-7 cells, tubular invaginations were significantly reduced or even absent in many cases. (C,D) Coexpression with N-WASP-VCA, a mutant that cannot interact with the Arp2/3 complex, failed to affect tubulation by SNX9. Likewise, coexpression of SNX9 with dynamin 2 K44A, a GTPase-deficient mutant, abolished the antagonistic effect of dynamin 2 on tubulation. (E-H) Representative pictures from various combinations of SNX9 or SNX9-SH3 with dynamin2 or N-WASP. Scale bars: 30 µm. (I) Quantitative analysis of tubule length in various experimental schemes. The length of tubules was measured manually using a MetaMorph software. Three rectangular areas (20x20 µm) near the periphery of each cell were drawn, and within these areas, the tubules that were well separated from other tubules were selected and their lengths were measured in the x-y dimension. Three independent experiments were performed and the data were pooled and normalized against those of full-length SNX9 with mRFP empty vector. Error bar indicates s.d. n=50 cells. ^*P<0.05, significantly different from SNX9-GFP or -mRFP control or each other, ANOVA and Tukey's HSD post hoc test for several different groups and Student's t-test for two different groups.