Fig. 4.

c-Rel O-GlcNAcylation is increased and Treg numbers are reduced in diabetic mice. (A) Splenocytes from normal or diabetic NOD mice were isolated and stained with anti-CD4 and anti-FOXP3 antibodies and analyzed by Flow Cytometry (n = 6) (B) Splenocytes from Control or C57BL/6 mice treated with STZ for 4 weeks (left) or 9 weeks (right) were isolated and stained as in (A). (C) Splenocytes were isolated from control or 9 weeks STZ-treated C57BL/6 mice were lysed, and c-Rel was immunoprecipitated from 25 to 30 × 10⁶ cells. The O-GlcNAcylation state of c-Rel was probed using the anti-O-GlcNAc antibody RL2 and compared with measured mouse blood sugar: Mouse 1 = 130 mg/dL, Mouse 2 = 128 mg/dL, Mouse 3 = 157 mg/dL, Mouse 4 = 144 mg/dL, Mouse 5 = 509 mg/dL, Mouse 6 = 565 mg/dL, Mouse 7 = 531 mg/dL, Mouse 8 = 520 mg/dL, Mouse 9 = 479 mg/dL, Mouse 10 = 151 mg/dL, Mouse 11 = 129 mg/dL, Mouse 12 = 522 mg/dL, Mouse 13 = 511 mg/dL and Mouse 14 = 408 mg/dL. Bottom panel shows total c-Rel in the immunoprecipitates. ns—non specific. (D) Authenticity of anti-c-Rel antibody was validated using total lysates of control and c-Rel KO fibroblasts.