Figure 4: Med23 ^−/− T cells are hyper-responsive to TCR stimulation.

Flow cytometry analysis of CD25 (a) and CD69 (b) expression on naïve T cells. Purified naïve T cells were unstimulated or stimulated with anti-CD3 (1 μg ml⁻¹) and anti-CD28 (1 μg ml⁻¹) antibodies for 16 h. (c) T-cell proliferation was measured by carboxyfluorescein succinimidyl ester (CFSE) dilution. Purified naïve T cells were stained with CFSE and stimulated using anti-CD3 (1 μg ml⁻¹) and anti-CD28 (1 μg ml⁻¹) antibodies for 48 h before fluorescence-activated cell sorting analysis. (d) IFNγ production from naïve T cells was analysed by flow cytometry. Purified naïve T cells were stimulated with anti-CD3 (1 μg ml⁻¹) and anti-CD28 (1 μg ml⁻¹) antibodies and restimulated with PMA and ionomycin for 4 h before intracellular staining. (e) Flow cytometry-sorted naive T cells were stimulated with anti-CD3 and anti-CD28 antibodies at various antibody concentrations and stimulation timepoints, and the expression of the surface marker CD69 was analysed by flow cytometry (n=3; *P<0.05 and **P<0.01 by Student’s t-test; NS, no significance). (f) Naive CD4⁺ T cells from WT and Med23^−/− mice were labelled with biotinylated anti-TCR (10 mg ml⁻¹) and anti-CD4 (10 mg ml⁻¹) antibodies and then cross-linked with streptavidin for the indicated times or left untreated (0 min). Total lysates from these cells were subjected to SDS-polyacrylamide gel electrophoresis and analysed with antibodies against the tyrosine-phosphorylated form of Erk1/2, Plcγ1, Src, Zap70/Syk, Jnk or P38. The expression of total Plcγ1 served as a loading control. Error bars indicate s.e.m. All results are representative of or combined from three independent experiments. All naïve T cells were sorted by flow cytometry based on high expression of CD62L and low expression of CD44.