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Figure 2. Membrane-targeted ILK increases Rac-1 activity in CCL39 fibroblasts. Activation of Rac-1 was measured in a GST-CRIB (PAK3) pull-down assay on (A) exponentially growing ILK-GFP and ILK-GFP-F expressing attached cells (Att), cells maintained in suspension in serum-free medium for 15 min (Sus) or cells plated on fibronectin-coated dishes (10 µg/ml) for 10 min (FN). B) Active Rac-1 was measured in CCL39 fibroblasts expressing ILK-GFP-F under control of a tetracycline-sensitive promoter in serum-deprived nontreated (control) or tetracycline-induced cells (1 µg/ml, 20 h) (+Tet) after adhesion (10 min) to fibronectin (FN) or stimulation or adherent cells with 10 ng/ml platelet-derived growth factor. ILK-GFP-F expression was monitored by Western blot analysis using anti-V5 Ab (left panel). Immunoblots were quantified and results were expressed as fold increase relative to control. Results represent mean values (±SEM) from 3 independent experiments.
