Fig. 2. FAK phosphorylation and stress fiber formation in cells plated on pLL. (A) Attachment and spreading after 3 hours. Mesangial cells were serum deprived for 24 hours and then non-enzymatically lifted and re-plated on either gelatin-coated (left) or pLL-coated (right) glass slides. After 3 hours, the cells were fixed and stained for F-actin (red, top panels) and vinculin (green, middle panels). Merged images are shown at the bottom. (B) Effect of TGF1 on FA and cytoskeletal structures. (a) Representative merged images of immunostaining for F-actin (red) and phospho-Y397 FAK (green) with cells re-plated on gelatin (top panels) or pLL (bottom panels) for 6 hours, followed by treatment with TGF1 (right panels) or vehicle (left panels) for 30 minutes. Bar, 20 µm. (b) Zoomed-in images of the FAs, shown next to their respective images, were obtained, using Photoshop 7.0 software (Adobe), to demonstrate colocalization (yellow). Bar, 2 µm. (c) Colocalizing pixels were selected by CoLocalization Express software (shown as dots in the image plots). The pixels of colocalization from four separate images were summed and are shown as a graph below the images. White bars, vehicle; black bars, TGF1. *P<0.05, compared to values in control conditions with vehicle. (C) Cells plated on gelatin or pLL for 4 hours were treated with TGF1 (1.0 ng/ml) or vehicle for 30 minutes and FAK phosphorylation at Y397 (top) or Y925 (middle) was analyzed by immunoblotting. (Graph, below) A representative set of blots and densitometoric analysis of four experiments for phospho-Y397-FAK (left) and phospho-Y925-FAK (right) over FAK expression are shown. White bars, vehicle; black bars, TGF1 treated. *P<0.05, compared to values on gelatin without TGF1; P<0.05, compared to values on gelatin without TGF1.