Xenbase Image ID: 173282

Fig. 1. Exogenous and endogenous Otx2 is phosphorylated. (A) Schematic structures of Myc-Otx2 and deletion constructs. HD, homeodomain; RD, repression domain; AD, activation domain; yellow box, Myc-tag; white star, Akt site; pink stars, Cdk sites. FL and deletion constructs are indicated by thick lines and positions of amino acid residues. (B) Western blotting of Myc-FL and Myc-δAD. TNT, in vitro translational products; embryo, lysate from Xenopus gastrula embryos expressing constructs as indicated. (C) Amino acid sequence of Otx2 from position 96 to 184. Numbers, position of amino acid; underline, repression domain; magenta, putative Cdk site; blue, putative Akt site; bold, possible phosphorylation sites identified in this study. (D) Western blotting of MycOtx2δAD constructs, WT, 3A and 4A. Lysates were treated with or without λ-PP. (E) IP-western assay for phospho-Akt sites. Myc-Otx2δAD was immunoprecipitated with anti-Myc antibody and subjected to western blotting with the anti-Phospho-Akt substrate antibody (upper) or anti-Myc antibody (lower). uninj. indicates uninjected control. The reasons why no clear band was detected in WT (lane 1) might be that: (i) phospho-Akt signals were dispersed in several shifted bands caused by phosphorylation at other sites; and (ii) phosphorylation at T115 was inhibited by phosphorylation at S116 or the other sites. (F) Western blotting of endogenous Otx2 (left) and exogenous Otx2FL (right) with anti-Otx2 antibodies. Lysates were treated with or without λ-PP. Apparent molecular masses (kDa) of the nascent and upper-most modified bands are indicated on the right (B,D-F). Calculated and apparent molecular masses of constructs (kDa): Myc-Otx2FL, 43 and 50; Myc-Otx2δAD, 32 and 40; Otx2FL, 31 and 33. White arrowhead indicates a band resistant to λ-PP or incomplete digests. 3A, alanine mutant at S116, S132 and S158; 4A, alanine mutant at T115, S116, S132 and S158; blue arrowheads indicate nascent proteins; magenta arrowheads indicate modified proteins. The amount of injected mRNA (pg/embryo) was Myc-Otx2FL and Myc-Otx2δAD, 500; Otx2FL, 250. Antibodies used for western blotting (WB) or IP were as indicated. Image published in: Satou Y et al. (2018) Copyright © 2018. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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