Figure 3. FRNK does not act as a dominant negative in early Xenopus embryos.

(A–D) Optical sections of whole mount immunostained embryos injected with 1 ng GFP-FRNK at the two dorsal blastomeres at the four-cell stage. Embryos were stained with anti-GFP (A) and anti-P-Y397 (B). C is the merged image and D an intensity color coded image of the anti-P-Y397 signal. FRNK injected cells are indicated with red stars and control cells with white stars. FRNK expression fails to reduce the phosphorylation levels of endogenous FAK on tyrosine 397. (E–H) Same as A–D, but the embryos were stained with anti-GFP (E) and anti-P-Y576 (F). FRNK expressing cells display similar levels of phosphorylation on tyrosine 576 as neighboring control cells. (I–L) Confocal images of A6 Xenopus cells transfected with GFP-FRNK. Cells were stained with anti-GFP (I) and anti-P-Y397 (J). K is the merged image and L an intensity color coded image of the anti-P-Y397 signal. FRNK expression leads to reduction of the phosphorylation levels of FAK on tyrosine 397 at the focal adhesions. (M–P) Same as I–L but the cells were stained with anti-GFP (M) and anti-P-Y576 (N) antibodies. FRNK expression leads to downregulation of the endogenous phosphorylation levels of FAK on tyrosine 576 at the focal adhesions. (Q) Western blot analysis of control and injected gastrula stage embryos with 1 ng FRNK at the animal pole of both blastomeres of two cell stage embryos. FRNK expression fails to reduce endogenous FAK phosphorylation on tyrosine 397. FRNK expression was verified using a FAK antibody raised against the C-terminus of the protein. (R–S) Localization of P-Y397 FAK (R) and FRNK (S) in animal pole cells of stage 10 Xenopus embryos. P-Y397 FAK shows strong membrane localization while FRNK is primarily cytoplasmic in these cells. Scale bars: (A) 40 µm, (E) 30 µm, (I) 20 µm, (M) 20 µm, (R–S) 40 µm.