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. 2012 Aug 6;7(8):e42577. doi: 10.1371/journal.pone.0042577

Figure 5. The FERM domain leads to activation of endogenous FAK in a tyrosine 397 dependent manner.

Figure 5

HA-FERM and HA-FERM Y397F injected embryos in one blastomere at the animal pole of two cell stage embryos were processed for whole mount immunostaining using an HA antibody (green) to reveal expressing cells and the respective phospho-specific antibodies (red) as indicated. In each case individual signals for each secondary are shown in addition to a merged image and finally an intensity color coded image of the respective phospho-specific antibody signal. HA-FERM and HA-FERM Y397F injected cells are indicated with red stars and un-injected cells with white stars. (A–D) Levels of phosphorylated tyrosine 576 are elevated in HA-FERM overexpressing cells compared to controls. (E–H) Overexpression of HA-FERM Y397F has no effect on the endogenous levels of phosphorylated tyrosine 576. HA-FERM Y397F expressing cells have the same levels of phosphorylated endogenous FAK on tyrosine 576 with neighboring control cells. (I–L) Levels of phosphorylated tyrosine 861 are elevated in HA-FERM expressing cells compared to controls. (M–P) Overexpression of HA-FERM Y397F has no effect on the endogenous levels of phosphorylated tyrosine 861. HA-FERM Y397F expressing cells have the same levels of phosphorylated endogenous FAK on tyrosine 861 with neighboring control cells. (Q) Total lysates from HA-FERM injected gastrula stage embryos contain comparable levels of endogenous FAK as un-injected controls but elevated levels of phosphorylated FAK on tyrosines 397, 576 and 861. Blotting using the anti-P-Y397 antibody shows that the exogenously expressed FERM is heavily in trans phosphorylated on tyrosine 397 (2nd row). The intensity values from the densitometry analysis were normalized against total FAK and present the average increase in phosphorylation from three independent experiments (R–U) Confocal images of A6 Xenopus cells transfected with HA-FERM. Cells were stained with anti-HA (R) and anti-P-Y576 (S). T is the merged image and U an intensity color coded image of the anti-P-Y576 signal. Transfected cells are shown with red stars and controls with white stars. HA-FERM transfected cells show reduced levels of tyrosine 576 phosphorylation suggesting that FERM expression blocks FAK activation in these cells. Scale bars: (A) 40 µm, (E) 30 µm, (I) 20 µm, (M) 50 µm, (R) 20 µm.