Figure 2.

Flow cytometric and Western blotting analyses comparing the binding functionalities of the various forms of E-selectin proteins to KG1a cells. A, E-S6-IgG, E-S6, E-S2, E-S2-A28H, and E-S0 were tested for their ability to bind to ligands on KG1a cells. Mouse monoclonal anti-Strep antibody (followed by a fluorescently labeled antibody against anti-Strep) was used to detect the E-selectin protein bound to ligands on the surface of KG1a cells in the presence of calcium (green histogram) or EDTA (red histogram). Samples stained with only the secondary antibody are included as a control to determine secondary antibody specificity toward mouse monoclonal anti-Strep antibody (blue histogram). B and C, the percentages of KG1a cells bound to each of the E-selectin proteins and the geometric means of the fluorescence signals were determined from n = 3 independent experiments (n = 3; a, indicates significance compared with E-S6-IgG; b, indicates significance compared with E-S6; c, indicates significance compared with E-S2; and d, indicates significance compared with E-S2-A28H, p ≤ 0.05) and the means are depicted as mean ± S.E.M. in (B) and (C), respectively. D, Western blot analysis of E-selectin protein binding to immunoprecipitated PSGL-1 and CD44. KG1a lysates were prepared, and PSGL-1 and CD44 were immunoprecipitated and subjected to the Western blot analysis. The resulting blots (upper panel, CD44; lower panel, PSGL-1) were stained with 1 μg/ml of E-S6-IgG, E-S6, E-S2, or E-S0, as indicated in the figure, in the presence of calcium. Anti-strep mAb was used against bound E-selectin for subsequent chemiluminescence detection using HRP-conjugated anti-mouse IgG. Blots stained in the presence of EDTA to confirm binding specificity showed no binding activity (Fig. S4).