N-Glycan Branching Inhibits Pro-inflammatory TLR4 and TLR2 Activation in B Cells
(A, B, F, and G) TLR4- and TLR2-stimulated B cells were assessed by flow cytometry for proliferation by CFSE dilution after 2 days (A), CD69 expression after 1 day (B), surface expression of CD80/CD86 after 1–3 days (F), and MHCII after 3 days (G). Histograms in (A and B) represent highest agonist dose.
(C and D) Western blot analysis of phospho-NFκB (p65) (C) and total TRAF3 protein and phospho-TBK1 (D) in B cells stimulated for 2 days.
(E) TNFα and IL-10 secretion measure by ELISA from culture supernatants of stimulated B cells over 1–3 days.
Data shown are mean ± SEM of cells stimulated in triplicate (A, B, E, and F) and representative of n ≥ 3 experiments. Each symbol represents one mouse from three different experiments; horizontal line represents the mean; repeated-measures ANOVA with false discovery rate correction (Benjamini, Krieger, and Yekutieli) for multiple comparisons (G). NS, not significant; ∗p < 0.05. Divided (%), CFSE dilution by half or more. MFI, mean fluorescence intensity.