Fig. 5: FOXK1, but not FOXK2, is modified by O-GlcNAcylation.

a HEK293T cells were transfected with constructs expressing Myc-FOXK1 or Myc-FOXK2 in the presence of OGT WT or OGT catalytically dead (CD) mutant (D925A). Myc immunoprecipitation was performed, and levels of O-GlcNAcylation were detected using an anti-O-GlcNAc specific antibody (n = 2 independent experiments). b Immunoprecipitation of endogenous FOXK1 was performed on U2OS cell extracts following transfection with siRNA targeting OGT (siOGT) or non-target siRNA as a control (siNT) (n = 3 independent experiments). c Immunoprecipitation of endogenous FOXK1 and analysis of O-GlcNAcylation in IMR90 cells treated with either; OGA inhibitors (PUGNAc, Thiamet G (ThG)) or OGT inhibitors (OSMI-4) (n = 3 independent experiments). d Immunoprecipitation and analysis of endogenous FOXK1 O-GlcNAcylation in IMR90 cells treated with glucose free media or gradually supplemented with increasing concentrations of glucose (n = 3 independent experiments). e Immunoprecipitation and analysis of endogenous FOXK1 O-GlcNAcylation in IMR90 cells synchronized by contact inhibition and released at low density in fresh medium (n = 3 independent experiments). f Immuno-depletion and analysis of endogenous FOXK1 O-GlcNAcylation in IMR90 cells. Cellular extracts from IMR90 were used for FOXK1 immunoprecipitation. Eluted proteins were then incubated with WGA bound beads, and FOXK1 O-GlcNAcylation levels were analyzed by western-blotting (n = 3 independent experiments). g In vitro O-GlcNAcylation was performed on recombinant GST-FOXK1 or GST-FOXK2 with recombinant His-OGT-Flag. The reaction was stopped at different time points to detect protein O-GlcNAcylation levels (n = 3 independent experiments). h Left: recombinant FOXK1 fragments are schematically represented and numbered. Right: in vitro O-GlcNAcylation was performed on recombinant FOXK1 fragments to map the region containing residues modified by O-GlcNAc (n = 3 independent experiments). i Top; schematic representing the identification of O-GlcNAc sites on FOXK1 as determined by mass spectrometry (MS) analysis. Mutant FOXK1^7A contains seven threonine mutated to alanine, whereas mutant FOXK1^11A contains all the eleven sites mutated to alanine. Bottom; FOXK1 structure predicted by Alphafold. The region highlighted is expected to be unstructured. Right; Visual representation of this region with the position of residues targeted by O-GlcNAcylation are shown on the predicted protein structure. j Immunoprecipitation of Flag-tagged versions of FOXK1, FOXK1^7A, or FOXK1^11A from IMR90 cell extracts and detection of O-GlcNAcylation levels (n = 3 independent experiments). For (a–h, j), the western blots are representative of one of the three independent experiments. Source data are provided as a Source Data file.