Fig. 5. ARAP3-deficient cells display increased Arf6 and altered Rac activities. (A) NA1 and 52B3 cells were seeded into tissue culture dishes, and either left to grow or subjected to serum starvation for 16 hours as indicated. Cells were lysed and lysates used for Arf `pull-down' assays using GST-GGA beads as bait. (B) Cells were seeded and starved as in (A) and subsequently stimulated with PDGF or its vehicle for 1, 5 and 10 minutes prior to cell lysis. For ease of comparison, basal levels were adjusted to 1. The graphs shown in (A) and (B) represent pooled data (means ± s.d.) from four independent experiments all of which were carried out in duplicate. (C) NA1 and 52B3 cells were seeded in tissue culture dishes, treated as in (A), and used for Rac `pull-down' assays using GST PAK-CRIB immobilised on Sepharose beads as bait. The shown graph represents pooled data (means ± s.d.) from four independent experiments. (D) Cells were treated as in (B) and used for Rac `pull-down' assays; basal levels were again adjusted to 1. The shown graph represents (E) HA-tagged, N27Arf6 or L67Arf6 transfected PAE, NA1 or 52B3 cells (as indicated) were seeded onto pooled data (means ± s.d.) from five independent experiments. (E) HA-tagged, N27Arf6 or L67 Arf6 transfected PAE, NA1 or 52B3 cells (as indicated) onto glass coverslips and serum starved for 16 hours. Cells were fixed, stained using an antibody for Rac1 and visualised by confocal microscopy. Photographed slices were 0.8 µm thick. Bar, 20 µm.