Figure 1: Ndrg1 is upregulated by an anergic stimulus and maintained in the resting anergic state.

(a) Kinetic analysis of gene expression following an anergic stimulus using oligonucleotide microarray chips. Biotinylated RNA probes prepared from A.E7 cells stimulated with anti-TCR for 2, 4 and 6 h or stimulated for 18 h followed by 5 days resting, were hybridized to Affymetrix gene chips. The amounts of mRNA are represented as the fluorescence intensity (average difference of 16 signals per gene). (b) Immunoblot analysis of Ndrg1 in A.E7 cells and pre-activated CD4⁺ primary cells (from 5CC7⁺ TCR transgenic mice) stimulated with anti-TCR for various time points. Un, unstimulated. (c) Resting A.E7 cells (untreated) or A.E7 cells made anergic by anti-TCR treatment for 18 h and resting for 5 days (anergic) were analysed by immunoblotting of Ndrg1(left panel) or stimulated with anti-CD3 and anti-CD28 for 48 h, to measure IL-2 production (right panel). (d) A.E7 cells treated with anti-CD3 plus various reagents for 18 h were subject to immunoblot analysis of Ndrg1 (upper blot) or washed and rested for 5 days before being stimulated with anti-CD3 and CD28, to measure IL-2 production (lower graph). CsA, cyclosporine A; Go, Gö6976; PD, PD98059; SB, SB203580; Rapa, rapamycin. Actin or glyceraldehydes 3-phosphate dehydrogenase (GAPDH) was used as a loading control for the immunoblotting. Results are representative of one (a) or two independent experiments (b–d).