Fig. 5

Decreased activation of PI3K, Erk1/2 MAPK, and p38 MAPK signaling pathways in Gnaq^−/− Bregs. Isolated B cells from WT and Gnaq^−/− mice cultured in presence of SB203580 (p38 inhibitor) (2.65 μM), U0126 (Erk1/2 inhibitor) (26 μM), or LY294002 (PI3K inhibitor) (6.4 μM) for 1 h, then cells stimulated by LPS for 48 h, and PMA, ionomycin, and brefeldin A added for last 5 h of culture. After culture, cells stained with anti-mouse CD19 and intracellular staining with IL-10, followed by flow cytometry analysis (a, representative images; b, statistical analysis). c Splenic B cells purified from WT and Gnaq^−/− mice and stimulated with LPS for 0–10 min. Protein from cell lysates exacted for analysis of phospho-PI3K, PI3K, phospho-Erk1/2, Erk1/2, phospho-p38 MAPK, and p38 MAPK by western blot analysis with specific antibodies individually. GAPDH used as loading control. d Protein expression levels quantified with Image Lab software. Ratios of phosphor-specific proteins versus total proteins obtained. Results represent mean ± SD per group (n = 4–5 mice/group). Data representative of three independent experiments. Student’s t test analyzed statistical difference. *p < 0.05, ***p < 0.001, ***p < 0.001. Breg regulatory B cell, CON control, Erk1/2 extracellular regulated protein kinases 1/2, GAPDH glyceraldehyde 3-phosphate dehydrogenase, IL interleukin, LPS lipopolysaccharide, ns not significant, PI3K PI3 kinase, PIM PMA/ionomycin/brefeldin A