Figure 4

The effect of α6β4 crosslinking on EGF-mediated Rho activation. MDA-MB-231 cells were incubated with anti-β4 on ice, followed by control rabbit IgG (lanes 3, 5, 7 and 9) or rabbit anti-mouse IgG (lanes 4, 6, 8, and 10) at 37°C to crosslink α6β4 for 15 min (lanes 3–6) or 30 min (lanes 7–10) in the presence (lanes 5, 6, 9, and 10) or absence (lanes 3, 4, 7, and 8) of EGF (10 ng/ml). Rho activation was assayed using a Rho pull-down assay with GST-tagged Rhotekin Rho-binding domain on glutathione-agarose beads. Negative and positive controls were MDA-MB-231 cell extracts loaded for 30 min at 30°C with 1 mM GDP (lane 1) or 100 μM GTPγS (lane 2), respectively.