Cross-linking of IRp60 inhibits JAK2 and MAP kinase phosphorylation in response to IL-5 and eotaxin. Eosinophils that were treated with anti-IRp60 or control antibodies (isotype, anti-p58.2) followed by sheep anti-mouse F(ab′)₂ were washed and IL-5 (50 ng/mL; A-C) or eotaxin (10-100 ng/mL; D) was added for the indicated time points. In panel A, after the activation step, cells were lysed and precleared. Thereafter, IL-5R_β chain was immunoprecipitated (IP), analyzed by SDS-PAGE, transferred to a membrane, and blotted with anti-phospho JAK2 (IB). Data shown are representative of 1 of 2 experiments. In panels B and D, after the activation step, the cells were fixed, permeabilized, and stained with mouse anti-phospho p38 (p-p38) or rabbit anti-phospho ERK 1/2 (p-ERK) followed by FITC-labeled goat antimouse and Cy5-labeled goat antirabbit. The intracellular staining was assessed by FACS. Data are representative of 1 of 3 experiments. In panel C, after the activation step, cells were lysed and analyzed by SDS-PAGE, transferred to a membrane, and blotted against phospho ERK 1/2 (IB). NA (dark gray shading) indicates nonactivated; Iso (solid line), isotype-matched control antibody treatment; IRp60 (bold solid line), anti-IRp60 treatment; p58.2 (dashed line), epitope control antibody treatment; IL-5 (light gray shading, B), IL-5 treatment; and Etx (light gray shading, D), eotaxin treatment.