Figure 5: Med23 promotes the transcription of negative regulators of T-cell activation.

(a) Real-time reverse transcriptase-PCR assay for Egr1, Egr2, Klf2 and Foxp1 expression in freshly isolated or in vitro-stimulated naive CD4⁺ T cells. Purified naïve CD4⁺ T cells were unstimulated (fresh) or stimulated using plate-bound anti-CD3 (1 μg ml⁻¹) and anti-CD28 (1 μg ml⁻¹) antibodies (Egr1 and Egr2, n=7; Klf2 and Foxp1, n=6; * P<0.05, **P<0.01 and ***P<0.001 by Student’s t-test; NS, no significance). The relative transcription levels of target genes were normalized to Gapdh. Naïve T cells were sorted by flow cytometry based on high expression of CD62L and low expression of CD44. (b) Purified naïve CD4⁺ T cells were unstimulated (fresh) or in vitro stimulated and lysed for ChIP assays (Egr1 and Egr2, n=3; Klf2 and Foxp1, n=4; *P<0.05 by Student’s t-test; NS, no significance). (c) Ratio statistics for CD69 expression, as examined by flow cytometry, in stimulated naïve CD4⁺ T cells transfected with lentiviral constructs. Purified naïve CD4⁺ T cells transfected with vector, Egr2 or Klf2 lentiviral constructs were in vitro stimulated using plate-bound anti-CD3 (1 μg ml⁻¹) and anti-CD28 (1 μg ml⁻¹) antibodies and evaluated by fluorescence-activated cell sorting analysis (Vector and Egr2, n=8; Klf2, n=7; *P<0.05 and **P<0.01 by Student’s t-test). Error bars indicate s.e.m. All results are combined from at least three independent experiments.