Fig. 4
From: Differential Lyn-dependence of the SHIP1-deficient mast cell phenotype
Lyn/Ca2+/calcineurin-dependent activation of the NFκB pathway in Ag-triggered BMMCs. Wt, Lyn-/-, SHIP1-/-, and dko BMMCs were preloaded and starved overnight with 0.15 μg/ml IgE. a BMMCs were left untreated (-) or stimulated with Ag (200 ng/ml) for the indicated time points. Whole-cell lysates were subjected to WB analysis with antibodies against P-IKKα/β (top panel), P-IκBα (second panel from top), IκBα (third panel from top), and p85 (loading control, bottom panel). Densitometry was performed and relative expression levels are indicated under each band. b BMMCs were left unstimulated (con) or stimulated with Ag (20 ng/ml) for 90 min. The amounts of Nfkbia mRNA (left panel) and Tnfaip3 mRNA (right panel) were measured by RT-qPCR. A comparison of analyses of different independent cell cultures is depicted in Additional file 6: Figure S7. For the response to different Ag concentrations see Additional file 7: Figure S6A. c Wt BMMCs, pretreated with vehicle (DMSO) or 100 nM CsA for 30 min, were left unstimulated (-) or stimulated with Ag (20 ng/ml) for the indicated time points. Whole-cell lysates were subjected to WB analysis with antibodies against P-IκBα (top panel), IκBα (middle panel), and p85 (loading control, bottom panel). d IgE-loaded SHIP1-/- BMMCs, pretreated with vehicle (DMSO) or 100 nM CsA for 30 min, were left unstimulated (-) or stimulated with Ag (20 ng/ml) for the indicated time points. Whole-cell lysates were subjected to WB analysis with antibodies against P-IκBα (top panel), IκBα (middle panel), and p85 (loading control, bottom panel). Densitometry was performed and relative expression levels are indicated under each band. A statistical analysis of the CsA effects is shown in Additional file 7: Figure S6C & D. e SHIP1-/- BMMCs, pretreated with vehicle (DMSO) or 100 nM CsA for 30 min, were left unstimulated (con) or stimulated with Ag (20 ng/ml) for 90 min. The amounts of Nfkbia mRNA (left panel) and Tnfaip3 mRNA (right panel) were measured by RT-qPCR. Comparable results were obtained with cells from different BMMC cultures (n = 3 (a, b); n = 3 (c, d); n > 3 (e))