Syk is tyrosine phosphorylated in the process of complement-mediated phagocytosis. (A) Tyrosine phosphorylation of Syk by the treatment with serum-opsonized zymosan. Macrophage-like differentiated Day3-HL60 cells were incubated with zymosan particles that were treated with PBS, serum, IgG-removed serum, serum containing compstatin (10 μM), or the control peptide to compstatin (10 μM) for 15 or 30 minutes (top). Macrophage-like differentiated Day3-HL60 cells were stimulated with CR3-activating F(ab)2 antibody VIM12 (2 μg/mL; bottom). Cell lysates were immunoprecipitated (IP) with antiphosphotyrosine (anti-PY) monoAb and immunoblotting analysis was performed with anti-Syk polyAb. As to the whole cell lysates, immunoblotting analysis was also done with anti-Syk polyAb. (B) Accumulation of Syk around the phagocytosed zymosan. At 5 minutes after the onset of phagocytosis, Day3-HL60 cells were fixed and stained with anti-Syk polyAb and AlexaFluor 488–conjugated secondary antibody. The arrows indicate the phagocytosed zymosan (the bar indicates 10 μm). A 63 ×/1.4 NA oil objective was used to visualize the images. DIC indicates differential interference contrast.
