Immunodepletion of PSGL-1 from the WGA affinity purified HL-60 lysate inhibits binding of P-selectin transfectants, but not L-selectin transfectants. (A) Western blot analysis showing immunodepletion of PSGL-1 from the HL-60 lysate. Lane 1: intact WGA-purified HL-60 lysate (220 to 250-kD band corresponding to PSGL-1); lane 2: lysate after immunodepletion with KPL1/protein G-Sepharose; lane 3: lysate after mock immunodepletion (incubation with protein G-Sepharose alone). PSGL-1 was detected by probing the blot with KPL1 ascites (1:2,500), followed by HRP-conjugated goat anti-mouse IgG and development using enhanced chemiluminescence. (B) Adhesion of 300.19 P-selectin or 300.19 L-selectin transfectants (1 × 106/mL) to PSGL-1–depleted HL-60 lysates under flow conditions (0.64 dyne/cm2) after immunodepletion of PSGL-1. Mean ± SD of bound cells/mm2 in multiple fields from two independent experiments.
