Syk affects actin dynamics around the C3bi-mediated phagosomes. Macrophage-like differentiated Day3-HL60, DN-Syk/HL (clone 8), Syk-siRNA/HL (clone 6), control siRNA/HL (clone 4), or Flag-rescue–Syk/Syk-siRNA/HL cells were incubated with serum-opsonized zymosan for indicated times and treated for morphologic studies (A-B) and immunoblotting analysis (C). (A) The parental HL60 cells were washed twice with culture medium and incubated for an additional 5 minutes or 30 minutes. The cells were fixed, stained with anti-Syk polyAb followed by AlexaFluor 488–labeled secondary antibody (green) and AlexaFluor 594–labeled phalloidin (red), and observed with a confocal laser-scanning microscope. Zymosan-containing phagosomes surrounded by F-actin are presented. A 63 ×/1.4 NA oil objective was used to visualize the images. DIC indicates differential interference contrast. (B) Zymosan-containing phagosomes existing in Day3-HL60, DN-Syk/HL, Syk-siRNA/HL, control siRNA/HL, or Flag-rescue–Syk/Syk-siRNA/HL cells were counted and the percentage of the phagosomes surrounded by F-actin to total phagosomes was calculated. One hundred cells were examined. Mean values and SD of triplicate experiments are shown. The statistically significant difference was assessed by the Student t test; *P < .05. (C) The cell lysates were immunoprecipitated with anti-Syk monoAb, and coprecipitated PLCγ2 was detected by immunoblotting analysis with anti-PLCγ2 polyAb. The reprobing analysis was also done with anti-Syk polyAb.