SHP-1 negatively regulates IL-6–induced STAT3 activation. (A) CD4⁺ T cells (0.7 × 10⁶) purified from +/+, me/+, and me/me C57BL/6 mice were stimulated with IL-6 for the indicated times. Cells were assessed for STAT3 phosphorylation (Tyr705), STAT3, and β-actin protein expression by immunoblotting. Numbers represent relative band densities in arbitrary units. (B) Kinetics of IL-6–induced STAT3 phosphorylation presented in panel A. pSTAT3 band densities normalized to β-actin (left panel) and pSTAT3 band densities normalized to STAT3 (right panel). (C) CD4⁺ T cells (0.7 × 10⁶) purified from +/+ and me/+ mice were stimulated with IL-6 for 10 minutes and analyzed as described in panel A. (D) Quantitative RT-PCR analysis of STAT3 mRNA isolated from splenic CD4⁺ T cells of +/+, me/+, and me/me C57BL/6 mice. STAT3 mRNA expression levels were normalized to HPRT1 and levels for +/+ set to 1. Data are representative of 2 or 3 independent experiments with multiple mice for each genotype.