Figure 3: Robo4 blocks Rac-dependent protrusive activity through inhibition of Arf6.

(a) Lysates from HEK 293 cells expressing the indicated constructs, plated on fibronectin and mock or Slit2, were precipitated with GST–PBD and immunblotted with Rac antibodies (n = 3). (b) Lysates from CHO-K1 cells expressing αIIb-Robo4 were precipitated with GST–PBD and immunoblotted with Rac antibodies (n = 3). (c) HEK 293 cells expressing Robo4 and Rac^G12V or LacZ were subjected to spreading assays on fibronectin and mock or Slit2. (d, e) Lysates from HEK 293 cells expressing full-length Robo4 (d) and CHO-K1 cells expressing αIIb integrin–Robo4 cytoplasmic tail (e), plated on the indicated matrix, were precipitated with GST–GGA3 and immunoblotted with Arf6 antibodies (n = 3). (f) CHO-K1 cells stably expressing αIIb or αIIb integrin–Robo4 cytoplasmic tail were co-transfected with GFP and either an empty vector or GIT1–PBS, and were subjected to spreading assays on the indicated matrix. (g) HEK 293 cells expressing GFP and the indicated constructs were subjected to spreading assays on fibronectin and Mock or Slit2. Expression of Robo4 and ARNO was verified by immunoblotting (data not shown). (h) Lysates from HEK 293 cells expressing the indicated constructs, plated on fibronectin and mock or Slit2, were precipitated with GST–PBD and immunoblotted with Rac antibodies (n = 3). For spreading assays, the area of GFP-positive cells was determined using ImageJ software (n = 3, 150 cells per experiment). Expression of Robo4 constructs was verified by western blotting (data not shown). *P < 0.05; **P < 0.005. Error bars indicate the mean ± s.e.m. Full scans of blots are shown in Supplementary Information, Fig. S7. αIIb–Robo4 indicates αIIb integrin–Robo4.