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Fig. 3. Expression of Y397F-FAK/YCam decreases phosphorylation of endogenous and exogenous FAK-Tyr-576 at FAs. (A) Cells were lysed and site-specific FAK phosphorylation was analysed in western blots with anti-FAK[P-Tyr-576] antibody. The amounts of proteins were monitored by stripping and reblotting the membranes with anti-FAK antibody. Note the reduction in phosphorylation of endogenous and exogenous FAK at Tyr-576 in Y397F cells. (B) Quantification of the percentage of endogenous (125 kDa; upper panel) and exogenous (199 kDa; lower panel) phosphorylated FAK-Tyr-576/total FAK in control and transfected cells. Data represent 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-576 phosphorylation was analysed by immunocytochemistry using anti-FAK[P-Tyr-576] antibody. Transfected Y397F-FAK/YCam cells (lower panel) have lower FAK Tyr-576 phosphorylation at FAs compared to adjacent non-transfected cells and to transfected FAK/YCam cells (upper panel). Scale bar: 10 µm. (D) Scatter plots show colour and intensity distribution of pixels in a pair of images (YCam, green and P-Tyr-576, red), with lower P-Tyr-576 intensities in Y397F cells. Extracted high intensity pixels (outlined white ROIs) were used to form a new image of the cellular localisation of these pixels (white images in upper right corners). (E) Quantification using Pearson's coefficient reveals no difference in colocalisation of YCam and P-Tyr-576 signals in Y397F-FAK/YCam cells compared to FAK/YCam cells.
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