Figure 3.
N-Glycan Branching Promotes Activation Signaling via CD19 and the B Cell Receptor Independent of CD22
(A, B, and D–F) Flow cytometric analysis of anti-IgM F(ab’)2-induced proliferation by CFSE dilution after 2 days (A) and Ca2+ mobilization over 4 min (B), and ex vivo B cells for IgM surface expression (D), CD19 surface expression (E), and CD19 endocytosis (F). Histograms in (A) represent highest stimulation concentration, arrow in (B) indicates addition of 2.5 μg/mL anti-IgM F(ab’)2, and immunofluorescent images in (E) were acquired on an Amnis ImageStream Imaging Flow Cytometer. Endocytosis rate over 1.5 h was calculated by dividing the MFI of acid-washed cells by the MFI of FACS buffer-washed cells divided by 1.5 h (F).
(C) Western blot analysis of phospho-CD19, phospho-Syk, and phospho-PLCγ in B cells stimulated with 10 μg/mL anti-IgM F(ab’)2. Data shown are mean ± SEM of cells stimulated in triplicate (A and F) and representative of n ≥ 3 experiments. Each symbol represents one mouse, and horizontal line represents the mean (D and E). Repeated-measures ANOVA with false discovery rate correction (Benjamini, Krieger, and Yekutieli) for multiple comparisons (D and E) and unpaired two-tailed t test (F). NS, not significant; ∗∗∗∗'p < 0.0001. MFI, mean fluorescence intensity.