Fig. 2

BMMCs deficient for Lyn and SHIP1 produce enhanced levels of pro-inflammatory cytokines in a PI3K-dependent manner. Wt, Lyn-/-, SHIP1-/-, and dko BMMCs were preloaded and starved overnight with 0.15 μg/ml IgE. a, b BMMCs were left unstimulated (con) or stimulated with Ag (20 ng/ml) for 4 h. Secreted IL-6 (a) and TNF-α (b) were measured by ELISA. Each bar is the mean of triplicates ± SD. For the response to further Ag concentrations see Additional file 3: Figure S3A & B. c, d BMMCs were left unstimulated (con) or stimulated with 20 ng/ml of Ag for 90 min. The amounts of Il6 mRNA (c) and Tnf mRNA (d) were determined by RT-qPCR using the Pfaffl method. A comparison of analyses of different independent cell cultures is depicted in Additional file 4: Figure S4. For the response to further Ag concentrations see Additional file 3: Figure S3C & D. e 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-PKB (upper panel) and p85 (loading control, lower panel). Densitometry was performed and relative expression levels are indicated under each band. Comparable results were obtained with cells from different BMMC cultures (n > 3 (a-e))