Figure 5. Phosphorylation of Ndrg1 correlates with the blockade of anergy.

(a) Immunoprecipitated Ndrg1 or the whole-cell lysate (WCL) from A.E7 cells stimulated for 18 h, as indicated in the presence of 1 μM of MG132, was analysed by immunoblotting with α-pAktS or anti-Ndrg1. Some cells were stimulated in the absence of MG132. (b,c) A.E7 cells stimulated with the indicated reagents for 18 h and rested for additional 5 days were subject to immunoblotting with anti-Ndrg1 (b) or restimulating with anti-CD3 plus anti-CD28 to measure IL-2 production (c). The cells stimulated with anti-CD3 plus anti-CD28 along with or without LY294002 were rested in the presence of 10 U ml⁻¹ of IL-2. (d) Immunoblot analysis of Ndrg1 in anergic A.E7 cells treated with IL-2 (1,000 U ml⁻¹) or IL-2 plus LY294002 for 24 h in the presence or absence of MG132. Anergic A.E7 cells were generated by 18 h stimulation with anti-CD3 and 5 days resting in the absence of IL-2. (e,f) Anergic A.E7 cells as in d were either left untreated or treated with IL-2 (30 U ml⁻¹) for 10 days in the presence or absence of LY294002. Next, the cells were analysed by immunoblotting with anti-Ndrg1 (e) or stimulated with various concentrations of anti-CD3 plus anti-CD28 (f). LY, LY294002 (25 μM); pNdrg1, phosphorylated Ndrg1; Un, resting untreated (unanergic); An, anergic. Results are representative of two (c,d) or three (a,b,e,f) independent experiments; error bars, s.d.