Journal: Nature communications

Article Title: The mediator subunit Med23 contributes to controlling T-cell activation and prevents autoimmunity.

doi: 10.1038/ncomms6225

Figure Lengend Snippet: 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 þ Tcells. Purified naı¨ve CD4 þ Tcells were unstimulated (fresh) or stimulated using plate-bound anti-CD3 (1 mg ml 1) and anti-CD28 (1 mg ml 1) antibodies (Egr1 and Egr2, n ¼ 7; Klf2 and Foxp1, n ¼ 6; * Po0.05, **Po0.01 and ***Po0.001 by Student’s t-test; NS, no significance). The relative transcription levels of target genes were normalized to Gapdh. Naı¨ve T cells were sorted by flow cytometry based on high expression of CD62L and low expression of CD44. (b) Purified naı¨ve CD4 þ Tcells were unstimulated (fresh) or in vitro stimulated and lysed for ChIP assays (Egr1 and Egr2, n ¼ 3; Klf2 and Foxp1, n ¼ 4; *Po0.05 by Student’s t-test; NS, no significance). (c) Ratio statistics for CD69 expression, as examined by flow cytometry, in stimulated naı¨ve CD4 þ Tcells transfected with lentiviral constructs. Purified naı¨ve CD4 þ Tcells transfected with vector, Egr2 or Klf2 lentiviral constructs were in vitro stimulated using plate-bound anti-CD3 (1 mg ml 1) and anti-CD28 (1 mg ml 1) antibodies and evaluated by fluorescence-activated cell sorting analysis (Vector and Egr2, n ¼ 8; Klf2, n ¼ 7; *Po0.05 and **Po0.01 by Student’s t-test). Error bars indicate s.e.m. All results are combined from at least three independent experiments.

Article Snippet: The anti-Pol II (N-20) and normal rabbit IgG antibodies used in the ChIP assays (4mg per sample) were obtained from Santa Cruz Biotechnology.

Techniques: Activation Assay, Reverse Transcription, Expressing, Isolation, In Vitro, Cytometry, Transfection, Construct, Plasmid Preparation, FACS