TCR-signaling pathways are intact in SOCS3-deficient T cells. (A) Total LN T cells from wt (+/+) or Socs3^ΔLck/ΔLck (Δ/Δ) mice were stimulated with 10 μg/mL anti-CD3 for the indicated times and immunoblotted with antibodies specific for the indicated proteins. An antibody specific for ERK was used as a loading control. (B) LN T cells were either unstimulated or stimulated with 10 ng/mL IL-2 or anti-CD3 and immunoblotted with the indicated antibodies. Antibodies specific for ERK and STAT5 were used as loading controls. LN T cells were stimulated for 24 hours with anti-CD3, and the expression of IL-2 was measured by quantitative RT-PCR (C) and enzyme-linked immunosorbent assay (D). (E) CD8⁺ T cells were stimulated for 20 hours with anti-CD3 with or without anti-CD28 and analyzed by an intracellular FACS for IL-2 production. Numbers in quadrants refer to the percentage of cells that fall within that quadrant. (F) Purified LN CD8⁺ T cells were CFSE labeled and cultured for 3 days with 5 μg/mL anti-CD3 either alone or in the presence of 20 μg/mL anti–IL-2 antibody. Numbers in quadrants refer to the percentage of cells that fall within that quadrant. (G) LN T cells were stimulated with IL-2 (20 ng/mL) for the indicated times, and STAT5 phosphorylation was measured by immunoblotting. Data are representative of 2 to 3 independent experiments. P-ERK indicates phospho-ERK; NFATp, nuclear factor of activated T cells; P-JNK, phospho-Jun N-terminal kinase; P-Stat5, phospho-STAT5. Values shown in C and D are means (± SD) of triplicate measurements. *P < .05.