FIG. 3.
Quantitation of Rac ruffling and ARF6 protrusion formation. (A) HeLa cells were transfected with either plasmids encoding Rac1 alone or those encoding Rac1 and either ARF6(T27N), 1-6(T31N), or 1-6 (1:5 ratio). (B) HeLa cells were transfected with either ARF6-HA alone or with ARF6-HA and either ARF6(T27N), 1-6(T31N), or 1-6 (1:5 ratio). Cells were incubated for 30 min in the presence of AlF and fixed, and the overexpressed proteins were labeled by immunofluorescence. For each condition, over 500 transfected cells were counted, and the fraction of Rac- or ARF6-transfected cells that were ruffling or forming protrusions, respectively, was noted. For cells overexpressing only Rac1 or ARF6, the fraction of ruffling or protruding cells was normalized to 1.0, and the other conditions were then expressed as a fraction of 1.0. Data shown are the means and standard errors of three independent experiments.