Src family kinases and Syk are involved in CD44-mediated phagocytosis. (A-D) Binding and uptake of CD44 Ebabs by macrophages was assessed quantitatively by light microscopy using a 63 ×/0.90 liquid-immersion objective lens (Leica Microsystems). Digital images were acquired through a Qimaging Retiga EX camera (Qimaging, Burnaby, Canada) and processed using OpenLab 3.4 software (Improvision, Lexington, MA). This figure illustrates the high level of both adhesion (tethering) and engulfment (white arrows) of CD44 Ebabs in the control assay (A-B) without specific inhibitor, whereas CD44 Ebabs preincubated with 50 μM PP1 Src-kinase pharmacologic inhibitor (C-D) were tethered but not engulfed. (E-F) Histograms illustrate the efficiency of phagocytosis of RAW macrophages preincubated with 0 or 50 μM PP1 or PP2 and subsequently fed CD44 Ebabs as prey. *Different from 0 μM inhibitor, P < .05, mean ± SE of 4 separate experiments. (G-H) Confocal fluorescent and bright field images of RAW macrophages labeled with antiphosphotyrosine antibodies during phagocytosis of CD44 Ebabs. The solid line represents 10 μm. (I-J) Confocal fluorescent and bright field images of RAW cells transfected with Syk-GFP and presented with CD44 Ebabs. Solid line represents 10 μm. (K) Efficiency of phagocytosis of CD44 Ebabs preincubated with 0 or 50 μM Syk inhibitor piceatannol. *Different from 0 μM piceatannol; P < .05, mean ± SE of 4 separate experiments. (L) Efficiency of phagocytosis of CD44 Ebabs by RAW cells expressing GFP-tagged Syk (K396R) dominant-negative fusions compared with nonexpressing control cells. *Different from nontransfected controls; P < .01, mean ± SE of 4 separate experiments. (M) Syk phosphorylation is increased in response to antibody crosslinking of CD44 as determined by Western blot analysis of whole-cell lysates (top) and Syk immunoprecipitates (bottom). Images are representative of 3 independent experiments. IB indicates immunoblot; IP, immunoprecipitation.