Fig. 2. Expression of Y397F-FAK/YCam decreases endogenous FAK-Tyr-397 phosphorylation at FAs. (A) Control U87, FAK/YCam (FAK) and Y397F-FAK/YCam (Y397) cells were lysed and site-specific FAK phosphorylation was analysed on western blots using anti-FAK[P-Tyr-397] antibody. The amounts of proteins were monitored by stripping and reblotting the membranes with anti-FAK antibody. In Y397F cells, note the absence of phosphorylation of exogenous FAK-Tyr-397 (199 kDa) and the reduced phosphorylation of endogenous FAK-Tyr-397 (125 kDa). (B) Quantification of the percentage of endogenous FAK-P-Tyr-397/total FAK in control and transfected cells. Data are mean±s.e.m. (n=5); ^*P<0.05, paired t-test. (C) FAK/YCam- and Y397F-FAK/YCam-transfected cells plated for 2 days on Matrigel were fixed, permeabilized and FAK-Tyr-397 phosphorylation was analysed by immunocytochemistry. Y397F-FAK/YCam transfected cells (lower panel) display very low levels of FAK-Tyr-397 phosphorylation at FAs compared to adjacent non-transfected cells and to FAK/YCam-transfected cells (upper panel). Scale bar: 10 µm. (D) Analysis of the localisation pattern of YCam (green) and P-Tyr-397 (red) pixels in FAK/YCam- and Y397F-FAK/YCam-transfected cells. The scatter plots (lower left corners) show colour and intensity distributions of pixels from a pair of images (YCam and P-Tyr-397). By selecting a ROI (outlined in white), a group of pixels with high intensities was extracted to form a new image of the cellular localisation of these pixels (white images in upper right corners). (E) Quantification using Pearson's coefficient (which describes the extent of overlap between image pairs) reveals a significant reduction (^*P<0.05, paired t-test) of correlation in YCam and P-Tyr-397 images for Y397F-FAK/YCam-transfected cells compared to FAK/YCam-transfected cells.