FIGURE 3.

PH domain of GEP100 is involved in Sema3E-induced cell collapse. A, domain structure of GEP100 and schematic representation of chimeric protein encoding PH domain of GEP100 fused to GFP. IQ, IQ motif; Sec7, Sec7 domain; PH, pleckstrin homology domain; CC, coiled-coil domain. B, expression of GFP-GEP100 PH in COS-7 cells. C and D, COS-7 cells were transfected with Plexin-D1-expressing plasmids, RFP-CAAX, and GFP-GEP100 PH. The cells were serum-starved and subjected to collapse assay as described for Fig. 1B. GFP-negative cell collapsed upon Sema3E stimulation (arrow), whereas GFP-GEP100 PH-expressing cells did not collapse (arrowheads). E, expression of HA-tagged GEP100 constructs in COS-7 cells. ΔPH, mutant GEP100 lacking PH domain. F, GEP100 siRNA-transfected HUVECs were plated on fibronectin-coated glass coverslips. The cells were transfected with GEP100 wild type or GEP100 ΔPH, serum-starved, treated with 100 ng/ml Sema3E for 15 min, and stained for F-actin. The endothelial cells exhibiting collapse phenotype were scored as a percentage of the total number of cells. The graph represents the means ± S.E. of three independent experiments. ns, no significant difference; **, p < 0.01. Scale bars, 20 μm.