Fig. 6

Lysine 220 is essential for in vivo Ilk function. (A) Phosphorylation of Akt/PKB at ser 473 and GSK-3β at ser 9 is not altered in loc/ilk mutant embryos. Whole embryo lysates of wt sibling embryos and loc/ilk mutant embryos at 3 dpf show identical amounts of phosphorylated Akt/PKB and GSK-3β by Western blot analysis using an anti-phospho Akt/PKB ser 473 or an anti-phospho GSK-3β ser 9 antibody. Western blotting with a non-phospho specific Akt/PKB antibody using the same lysates shows identical levels of Akt/PKB protein present in both samples. Anti-GAPDH was used as an additional loading control. (B) Relative expression levels of Ilk in embryonic extracts of uninjected (control) or gfp–ilk, gfp–ilkK220M and gfp–ilkK220A mRNA injected embryos. (C–F) An uninjected control embryo with a weak and non-specific autofluorescence (C). Embryos injected with synthetic mRNA encoding wt GFP–Ilk (D), GFP–Ilk E359K (E) or GFP–Ilk K220M (F) all show a normal localization of the GFP–Ilk protein to the MTJs located at the somite boundary. (G–J) Muscle detachments are obvious in loc/ilk mutant embryos (G). The phenotypes including muscle detachments of loc/ilk mutant embryos were rescued by injection with synthetic mRNA encoding wt Ilk (H) or Ilk E359K (I). No rescue was observed in loc/ilk mutant embryos injected with mRNA encoding Ilk K220M (J).