Siglec-E is a negative regulator of CD11b-dependent phosphorylation of Syk and p38 MAP kinase. (A) Increased phosphorylation of Syk and p38 MAP kinase in siglec-E^−/− cells following adhesion to fibrinogen. WT and siglec-E^−/− bone marrow cells were plated at 37°C under the different conditions and times indicated, and whole cell lysates were immunoblotted with antibodies against phopho-Y³¹⁷Syk, total Syk, phospho-p38, and total p38. As a negative control, cells were treated with the Src family kinase inhibitor PP2 (20 µM). As a positive control, cells were treated with freshly prepared pervanadate (PV) to inhibit cellular tyrosine phosphatases. Phospho-Syk and phospho-p38 signals for each sample were normalized to their respective total Syk or total p38 signals. Data are representative of 3 independent experiments. Bar charts show densitometry expressed as means ± SEM; n = 3 per group; and *P < .05, paired t test compared with WT control. (B) Siglec-E binds fibrinogen in a sialic acid–dependent manner. Siglec-E-Fc precomplexes were incubated on fibrinogen pretreated with or without sialidase. Bar charts show densitometry expressed as means ± SEM; n = 3 per group; and *P < .05, paired t test compared with untreated membranes. (C) Sialidase treatment of fibrinogen reverses siglec-E–dependent inhibition of phopho-Y³¹⁷Syk. Wells were coated with fibrinogen or IgG/anti-IgG complexes and either left untreated (No buffer) or were treated with V. cholerae sialidase (Sialidase) or sialidase buffer alone (Buffer) for 60 minutes. After blocking with 10% fetal calf serum, bone marrow cells were allowed to adhere for 20 minutes. Lysates were immunoblotted with antibodies to phospho-Y³¹⁷Syk antibody and total Syk. Bar charts show densitometry expressed as means ± SEM; n = 4 per group; and *P < .05, paired t test compared with WT control.