Fig. 3

Mature B cells lacking Ogt do not activate after BCR cross-linking. a Flow cytometric analysis showing the frequency of Annexin V⁺ cells in Ctrl and B-KO splenic B cells stimulated with anti-IgM (10 µg ml⁻¹) or LPS (2 µg ml⁻¹) for 24 h. b Immunoblot showing the levels of CD79a, Lyn and Syk activation in Ctrl and B-KO splenic B cells after anti-IgM stimulation. c Immunoblot showing the nuclear translocation of NF-κB subunits in Ctrl and B-KO splenic B cells after anti-IgM stimulation. Lamin B: nuclear protein loading control. d Immunoblot showing the activation of PI3K and Akt in Ctrl and B-KO splenic B cells after anti-IgM stimulation. e Flow cytometric analysis showing the levels of CD69 and CD86 in Ctrl and B-KO splenic B cells stimulated with anti-IgM or LPS for 24 h. Bar graphs on the right show the relative mean fluorescent intensity of CD69 (top panel) or CD86 (bottom panel) normalized by that of Ctrl B cells. f Flow cytometric analysis showing the frequency of BrdU⁺ cells in Ctrl and B-KO splenic B cells stimulated with anti-IgM or LPS for 48 h. g Immunoblot showing the phosphorylation and total levels of Lyn, Syk and CD79a after rBAFF (200 ng ml⁻¹) treatment in Ctrl and B-KO splenic B cells. The representative data from one of at least three independent experiments (two independent experiments in b) are shown. Quantification results shown in the right panel in a, e and f are the mean ± s.e.m. (n = 3). *P < 0.05, **P < 0.01 (two-tailed unpaired t test). In b, c, d and g, quantification of band intensity is relative to Lamin B (c) or each corresponding total protein (b, d and g) as indicated. N.S., not significant