Comparison of mAbs 2PH1, 4RA10, and 4RB12 against PSGL-1.

(A) The total cell lysate from surface biotinylated 32Dcl3 cells was subjected to immunoprecipitations with the mAb 2PH1, 4RA10, and 4RB12 against mouse PSGL-1 (α-PSGL-1) as indicated. The mAb 2PH1 described by Borges et al19 served as control. The first lane (Co) is the negative control with a mAb against CD44. Precipitated antigens were blotted and the filter was probed with horseradish peroxidase–conjugated streptavidin. The 230/130-kd double band of PSGL-1 is indicated by arrows. Molecular weight markers (kd) are indicated on the left, and the front (F) of the gel is marked. (B) Inhibition of P-selectin–IgG binding to the mouse T-cell clone 4G3, as analyzed by flow cytometry. Cells were incubated either with P-selectin–IgG (P-Sel-IgG) or as negative control with human IgG (hIgG) and binding was detected with a fluorescence-labeled secondary antibody. To test the inhibitory activity of the anti–PSGL-1 antibodies, cells were first incubated with the anti–PSGL-1 mAb 4RA10, 2PH1, or 4RB12 (as indicated) and subsequently incubated with P-selectin–IgG fusion proteins (P-Sel-IgG). Binding of P-selectin–IgG was detected with a fluorescence-labeled secondary antibody and staining was determined by FACS analysis. For positive controls, P-selectin–IgG was analyzed without preincubation with an anti–PSGL-1 mAb; for negative controls, cells were incubated with human IgG (hIgG) and the secondary reagent.