(A, B) MDA-MB-468 cells were depleted of p33 Rsu-1 (siRNA Rsu) or p29 Rsu-1 (siRNA RsuJ) for 72 h. (A) siRNA-treated cells (5 × 104) were seeded on matrigel-coated membranes in Boyden chambers. At 20 h post seeding the cells migrating through the membrane were fixed, stained and enumerated (lower panels). The percentage of cells migrating was normalized to that of control siRNA-treated cells (siRNA c) which was set at 1.0. Upper panels: Verification of reduced Rsu-1 expression after siRNA treatment. (B) siRNA-treated cells were harvested and the level of Rac-GTP was determined by binding to the GST-Rac-binding domain of Pak1 (GST-PBD) (upper panel). (C) Cos-1 cells were transfected with an empty vector or a vector encoding p33 Rsu-1. At 72 h post transfection the cells lysates were prepared from cells with or without EGF stimulation (100 ng/ml, 7 min), and the level of Rac-GTP was determined (upper panel). Five percent of the total cell lysate was included as control for cellular levels of Rac (B, C; lower panels).