Figure 6.

Rac1 is activated when cells are plated on collagen I. (A) NMuMG/E10 cells were extracted 3 to 24 h after plating on noncoated or collagen I-coated dishes. Lysates (1 mg of protein) were incubated with GST-CRIB–coupled beads and resolved by SDS-PAGE. Rac1-GTP and total Rac1 were detected by Rac1 immunoblots. (B) Rac1-GTP was quantified and normalized to total Rac1. Each pull-down experiment was repeated two times. (C) NMuMG/E10 cells were infected with retrovirus encoding the neomycin-resistance gene (MOCK), RacN17-GFP, or RacV12-GFP. Three hours after seeding on noncoated or collagen I-coated dishes, protein was extracted and pull-down assays performed as in A. (D) RIPA extracts of NMuMG/E10 cells expressing the neomycin-resistance gene (MOCK), RacN17-GFP, or RacV12-GFP were immunoblotted for phospho-JNK (p-JNK; Thr183/Thy185) and total JNK1. Cells treated with anisomycin (Aniso; 1 μg/ml) to activate JNK were used as a control to indicate the phosphorylated forms of JNK (lane 7).