Fig. 3. Adhesion to collagen I increases the GTP-loading of endogenous Rac1 and transfected myc-tagged Rac1 in primary human fibroblasts. (A) Primary human fibroblasts were incubated in suspension for 2.5 hours and then aliquots of these cells were adhered to collagen I (50 µg/ml) for 15, 30 or 45 minutes as indicated. The amount of Rac1 GTP-loading was compared in lysates from suspended and adhered cells. The levels of Rac1 detected by western blot in the GST-PAK pull-down assay (top panel) and the lysate (bottom panel) are shown. (B) The average fold change in endogenous Rac1 GTP-loading±s.e.m. from three trials is shown in the bar graph. The level of Rac1-GTP in suspended cells was normalized to 1. (C) Primary human fibroblasts were transiently transfected with myc-tagged wild-type Rac1 and the amount of Rac1 GTP-loading was compared in lysates from suspended cells and cells adhered to collagen I for 15 and 30 minutes. The average fold change in Rac1-GTP loading±s.e.m. from three trials is shown in the bar graph. The level of Rac1-GTP in the suspended transfected cells was normalized to 1. Significant differences are indicated (*).

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