Schematic workflow of FOXO3 protein separation, proteomic analysis, and O-GlcNAc-modified site identification. Schematic workflow of FOXO3 protein separation,

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Schematic workflow of FOXO3 protein separation, proteomic analysis, and O-GlcNAc-modified site identification. Schematic workflow of FOXO3 protein separation, proteomic analysis, and O-GlcNAc-modified site identification. A, Purification of FOXO3 from FOX-OV cells via pH-fractionation and IP, and the identification of O-GlcNAc sites in FOXO3 by MS. B, FOXO3 expression levels in transfected cells were determined by Western blot analysis. C, FOXO3 expression levels in Agilent 3100 OFFGEL fractionator-enriched samples were determined by Western blotting. D, Schematic diagram of seven identified novel O-GlcNAc-modified sites within FOXO3, determined from FOX-OV cells via MS. All presented data were quantified from three independent experiments. N, N-terminus; Forkhead, forkhead domain; L, nuclear localization sequences; E, nuclear export sequences; TAD, transactivation domain; C, C-terminus. Heon Shin et al. Cancer Res 2018;78:1214-1224 ©2018 by American Association for Cancer Research