Effect of ARF6 on spine development is partly blocked by dominant-negative Rac1. A, Cultured neurons were transfected at DIV 9 with ARF6-HA T157A plus EGFP, ARF6-HA T157A plus EGFP plus FLAG-Rac1 N17, or EGFP alone, and double-labeled at DIV 11 for EGFP, HA, and FLAG. Scale bar, 5 μm. B–D, Quantitation of the Rac1 N17-dependent inhibition of the ARF6 T157A effects on the linear densities of spines, filopodia, and total protrusions. EGFP alone (protrusion density, 54.44 ± 2.19; spine density, 35.72 ± 2.30; filopodia density, 18.72 ± 1.02; length, 2.73 ± 0.10; width, 0.80 ± 0.02); ARF6 T157A (protrusion density, 55.95 ± 1.69; spine density, 48.77 ± 1.82; filopodia density, 7.18 ± 0.84; length, 1.85 ± 0.07; width, 1.00 ± 0.02); ARF6 T157A + Rac1 N17 (protrusion density, 64.89 ± 2.58; spine density, 44.15 ± 2.12; filopodia density, 20.74 ± 1.56; length, 2.60 ± 0.07; width, 0.89 ± 0.03). E–H, Quantitation of the Rac1 N17-dependent blockade of the ARF6 T157A effects on the lengths and widths of protrusions. Data were collected from 800–100 protrusions on 18–26 dendrites of 14–21 neurons for each group in the two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA).