Fig. 1.

O-GlcNAc-cycling enzymes regulate FOXP3 stability in vitro. a, b Mean fluorescence intensity (MFI) of OGT (a) and O-GlcNAcylation (b) in CD4⁺CD25⁻naïve T cells, CD4⁺CD25⁺FOXP3⁺ Treg cells and corresponding Fluorescence Minus One (FMO) negative controls. c Treg cells isolated from wildtype mice were stimulated with or without anti-CD3/CD28 beads for 24 h ex vivo (n = 3). MFI of FOXP3 and O-GlcNAcylation was analyzed in CD4⁺CD25⁺ FOXP3⁺ Treg cells. d, e HEK 293 cells were transfected FOXP3 together with OGT or OGA (d) or treated with inhibitors of OGT (ST045849) or OGA (TMG) (e). FOXP3 O-GlcNAcylation was determined by immunoprecipitation followed by western blotting. f FOXP3 stability was determined by treatment of cycloheximide (CHX) in combination with MG132 with or without OGT overexpression, DMSO was used as a control for MG132. g–i FOXP3 stability was determined in the presence of OGT/OGA overexpression (g), TMG (h), or ST045849 (i). Data are shown as mean ± s.e.m. *p < 0.05 by unpaired student’s t-test