Figure 1: The Robo4 cytoplasmic tail suppresses fibronectin-induced protrusive activity.

(a) Schematic representation of cDNA constructs used in migration and spreading assays. TM, transmembrane domain. CC0 and CC2 are conserved cytoplasmic signalling motifs found in Robo family members. (b–d, f) HEK 293 cells expressing GFP and the indicated constructs were subjected to haptotaxis migration assays (b, f) or spreading assays (c, d) on fibronectin and mock or Slit2. Arrows indicate GFP-positive cells. (e) CHO-K1 cells stably expressing αIIb integrin or αIIb integrin–Robo4 cytoplasmic tail were subjected to spreading assays on fibronectin (Fn) or fibronectin and fibrinogen (Fn + Fgn). Cell area was determined using ImageJ software. For migration assays, GFP-positive cells on the underside of the filter were counted and migration on fibronectin or mock membranes was set at 100% (n = 3, in triplicate). For spreading assays, the area of GFP-positive cells was determined using ImageJ (n = 3, 150 cells per experiment). Expression of Robo4 constructs was verified by immunoblotting (data not shown). *P < 0.05; **P < 0.005. Error bars indicate the mean ± s.e.m. WT, wild type. Mock, a sham preparation of Slit2. αIIb–Robo4, αIIb integrin–Robo4. pCDNA3, empty vector.