Assessment of PIR-B with various signaling molecules at baseline and after eotaxin and LTB4 stimulation. Eosinophils were obtained from the spleens of CD2–IL-5Tg mice. Freshly isolated eosinophils were incubated with various concentrations of sodium orthovanadate (A). Thereafter, the cells were lysed, precleared, PIR-B immunoprecipitated (IP), analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a membrane, and blotted (IB) with antibodies to SHP-1, SHP-2, and PIR-B as a loading control. After eotaxin-2 stimulation (B), the eosinophils were lysed, precleared, IP, analyzed by SDS-PAGE, transferred to a membrane, and IB with antibodies to phospho-tyrosine (pTyr), SHP-1, and Hck. As a loading control, samples were also IB with anti–PIR-B. PIR-B–kinase complexes were assessed using an antibody-coated membrane. Densitometric analysis of phosphorylation patterns after eotaxin or LTB4 stimulation (C-G) is shown. Data are normalized to baseline phosphorylation status and are shown as the percentage change (increase or decrease) from unstimulated eosinophils. Data represent means plus or minus SD of n = 4; *P < .05; **P < .01; when comparing LTB4- and eotaxin-treated cells.
