Expression of Siglec-8 and Siglec-8L by eosinophils and transfected cell lines. Purified peripheral blood eosinophils (A) and HEK293T cells stably transfected with Siglec-8 or Siglec-8L (B) were analyzed by flow cytometry for Siglec-8 surface expression using murine monoclonal antibodies (2E2, 2C4, and/or 9G4). Histograms shown are representative of 3 to 4 experiments with identical results. Monoclonal reagents used as positive (CD18) and negative controls are also shown. (C) Western blot analysis of human peripheral blood eosinophils that were obtained from 2 different atopic donors. Lysates from these cells, as well as from Siglec-8L–transfected HEK293T cells, were generated. Equal amounts of protein from each sample were analyzed by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with sheep antihuman polyclonal Siglec-8 Ab or His₆ Ab. Wild-type HEK293T cells and Jurkat T cells were used as negative controls. Data are representative of 3 different experiments with similar results. Marker indicates molecular weight standard markers.