FIGURE 5:

Weak Syk recruitment in RBL-Lyn^KO cells is a result of incomplete FcεRIγ ITAM phosphorylation. (A, B) Confocal images of RBL-Lyn/γ^dKO cells expressing both Syk-mNG (green) and an FcεRIγ mutant lacking either the N-terminal (γ-FY; A) or C-terminal (γ-YF; B) tyrosine. Cells were incubated overnight with IgE-AF647 (magenta) and activated with 0.1 µg/ml DNP₂₅-BSA. Recruitment of Syk-mNG was observed for cells expressing γ-FY (n = 65 cells) but not γ-YF (n = 83 cells). See also Supplemental Figure S1. Scale bars are 5 µm. (C) Quantification of Syk cluster formation over time. Time series of Syk-mNG clustering was acquired using TIRF. Each cell was normalized to its maximum cluster number; mean and SD of cluster values between cells; RBL-WT n = 7, RBL-Lyn^KO n = 5, RBL-Lyn/γ^dKO(γ-FY) n = 5. * P < 0.05, ** P < 0.01, two-tailed ANOVA with Tukey’s multiple comparison test. All other differences were nonsignificant.