E-selectin engagement of PSGL-1 or CD44 activates SFKs in a lipid raft–dependent manner and then sequentially activates Syk, Btk, and p38. (A) WT leukocytes were rotated on E-selectin–IgM in the presence or absence of EDTA (ethylenediaminetetraacetic acid) or on control CD45-IgM for 5 minutes. Lysates were immunoprecipitated (IP) with antibody to the indicated SFK and then Western blotted (WB) with the same antibody or with anti–phospho-SFK (Y416). (B) Leukocytes of the indicated genotype were incubated as in panel A. Lysates were probed with antibody that recognizes all SFKs or with anti–phospho-SFK (Y416). (C) WT leukocytes treated with the indicated agent were incubated as in panel A. Lysates were probed with antibody that recognizes all SFKs or with anti–phospho-SFK (Y416). (D) WT leukocytes treated with the indicated agent or leukocytes of the indicated genotype were incubated as in panel A. Lysates were probed with antibody that recognizes all SFKs or with anti–phospho-SFK (Y416). (E) WT leukocytes treated with the indicated agent were incubated as in panel A. Lysates were probed with antibody to Syk or to phospho-Syk. (F) Lysates were Western blotted with antibody to Syk or to phospho-Syk. (G) Lysates were immunoprecipitated with antibody to Btk and then Western blotted with the same antibody or with antiphosphotyrosine antibody. (H) Lysates were probed with antibody to p38 or to phospho-p38. The E-selectin density was 400 sites/μm² for PSGL-1^−/−/CD44^−/− leukocytes and 200 sites/μm² for leukocytes of all other genotypes. The data are representative of at least 3 independent experiments.