Fig. 2

O-GlcNAcylation stabilizes the FOXP3 protein in Treg cells. a–d Treg cells isolated from Ubc-CreER⁺Ogt^fl/Y mice were treated with 4-OHT for 3⁻day ex vivo, ethanol was used as the control, n = 4 each group. MFI of FOXP3 in CD4⁺FOXP3⁺ Treg cells was analyzed in a and quantified in b. Representative flow cytometry of CD4⁺ FOXP3⁺Treg cells was plotted in c and the frequencies of Treg cells were shown in d. e FOXP3 or FOXP3-5A were transfected in HEK 293 cells with or without OGT. FOXP3 O-GlcNAcylation was determined by immunoprecipitation followed by Western blotting. f FOXP3 and FOXP3-5A stability was determined by treatment of CHX in the presence of OGT overexpression. g, h CD4⁺CD25⁻ naïve T cells isolated from wildtype mice were infected with retroviruses expressing FOXP3 or FOXP3-5A in the presence of anti-CD3/CD28 beads (n = 4). MFI of FOXP3 was analyzed (g) and quantified (h) in CD4⁺FOXP3⁺ Treg cells. Data are shown as mean ± s.e.m. *p < 0.05; **p < 0.01 by unpaired student’s t-test (b, d) and paired student’s t-test (h)