PSGL-1 and CD44 require core 1-derived O-glycans and sialic acids to initiate signaling. (A) WT or C1galt1−/− neutrophils were incubated on immobilized F(ab′)2 fragments of isotype control, anti–PSGL-1, or anti-CD44 mAb. Lysates were probed by Western blotting with antibodies to phospho-SFK (p-SFK), total SFK, phospho-p38 (p-p38), or total p38. (B) WT neutrophils were incubated with buffer or neuraminidase (sialidase) and then incubated on immobilized F(ab′)2 fragments of isotype control, anti–PSGL-1, or anti-CD44 mAb. Lysates were probed by Western blotting with antibodies to p-SFK, total SFK, p-p38, or total p38. Results are representative of three experiments.