FIGURE 3.

Arf5 but not Arf6 restores normal spreading in BRAG2-depleted cells. A, surface area of spread cells was measured for control (mock-depleted) or BRAG2-depleted cells as in Fig. 2. B, BRAG2-depleted cells were transfected with plasmids encoding rapid-cycling mutants of either Arf5-HA (Arf5-RC) or Arf6-HA (Arf6-RC), and spreading was measured as described in A. C, neither rapid cycling Arf5 nor Arf6 affects spreading in control, non-BRAG2-depleted cells. Surface areas of spread cells were measured as indicated in the legend to Fig. 2. D, mean surface areas ± S.E. were calculated for each condition from three replicate (n = 3) experiments. A one-way parametric analysis of variance (with Bonferroni's multiple comparison test) was performed to demonstrate statistical significance, where p < 0.0005 are denoted by ***; and p < 0.00001 are denoted by ****. E, relative binding of wild-type Arf5-HA or Arf5-T161A-HA rapid cycling mutant to GST-GGA3 was determined in a pulldown assay. F, a representative immunoblot showing the extent of BRAG2 depletion and relative expression of rapid-cycling Arf5 and Arf6. NS, not significant.