Fig. 1. The DHR-1 domain is required for DOCK180-mediated cell elongation and motility.

a) Serum-starved LR73 cells that had been transfected with the indicated plasmids were detached and plated on fibronectin-coated cover slips for 2 h in the absence of serum prior to fixing. Cells in the left panels were stained with anti-DOCK180 (H-4), while the panels on the right represent an overlay of the anti-DOCK180, rhodamine-phalloidin and DAPI stains. Cells were photographed at a 60X magnification. b) Expression levels of the transfected proteins were analyzed by immunoblotting of cell lysates with anti-DOCK180 (C-19) and anti-Myc antibodies, as indicated. c, d) Serum-starved LR73 cells that had been transfected with a GFP plasmid together with the indicated plasmids were detached and placed in the upper compartment of a Boyden-chamber. In the haptotactic migration assay (c), the cells were allowed to migrate for 4 h towards fibronectin. In the chemotactic assay (d), the top and bottom sides of the membrane were precoated with fibronectin and the cells were allowed to migrate for 4 h towards 10% serum as a chemoattractant in the lower chamber. Cells were fixed and stained with DAPI. GFP/DAPI double positive cells that had migrated across the membrane were counted from photographs taken at a 40X magnification. The experiments described above were performed independently three times in duplicate. Data are shown as mean +/- SD. *, P < 0.001; one-way ANOVA. e) Expression levels of the transfected proteins were analyzed by immunoblotting of cell lysates with anti-DOCK180 (C-19) and anti-Myc antibodies, as indicated.