Neither DN-Syk nor Syk-siRNA affects the expression of the complement receptor CR3. DN-Syk was transferred into HL60 cells and stable mutant clones (DN-Syk/HL, cl3 and cl8) were isolated. Syk-siRNA or control siRNA was also transferred into HL60 cells and stable mutant clones (Syk-siRNA/HL, cl2 and cl6; control siRNA/HL, cl1 and cl4) were isolated. Flag-rescue–Syk was transferred into Syk-siRNA/HL cl6 and stable mutant was isolated. Protein expression was examined in these mutant clones and parental HL60 cells. (A) Expression of transferred Flag-tagged DN-Syk protein in DN-Syk/HL (cl3 and cl8) cells was confirmed by immunoblotting analysis using anti-Syk polyAb and anti-Flag monoAb (top panel). Macrophage-like differentiated Day3-HL60 and DN-Syk/HL cells were incubated with serum-treated zymosan. Cell lysates were immunoprecipitated with antiphosphotyrosine 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 (bottom panel). (B) Expression of endogenous Syk was examined in Syk-siRNA/HL (cl2 and cl6), control siRNA/HL (cl1 and cl4), and HL60 cells by immunoblotting analysis using anti-Syk polyAb (top panel). Expression of Flag-rescue–Syk was examined by immunoblotting analysis using anti-Syk polyAb and anti-Flag monoAb (bottom panel). (C) Cell-surface expression of CR3 (integrin αMβ2) on macrophage-like differentiated Day3-HL60, DN-Syk/HL (cl3 and cl8), Syk-siRNA/HL (cl2 and cl6), control siRNA/HL (cl1 and cl4), and Flag-rescue–Syk/Syk-siRNA/HL cells were analyzed by flow cytometry with anti-CR3 monoAb (thick line) or with control mouse IgG (thin line).