Fig. 1

Reduced degranulation of Lyn-/- BMMCs despite abolished SHIP1 tyrosine phosphorylation. Wt and Lyn-/- BMMCs were preloaded and starved overnight with 0.15 μg/ml IgE. a BMMCs were left untreated (-) or stimulated with 200 ng/ml Ag (DNP-HSA) for the indicated time points. Whole-cell lysates were subjected to WB analysis with antibodies against phospho-tyrosine (P-Tyr; upper panel) and p85 (loading control, lower panel). Comparable results were obtained with 20 ng/ml Ag (see Fig. 3c). b Ca²⁺ mobilization was measured in wt and Lyn-/- BMMCs for 4 min by flow cytometry using the Ca²⁺-sensitive fluorescent dyes fluo-3 (Ca²⁺ b (bound)) and fura red (Ca²⁺ ub (unbound)). Steady-state fluorescence was measured for 1 min before BMMCs were stimulated with Ag (20 ng/ml). The arrow marks the time point of stimulus addition. For other Ag concentrations see Additional file 1: Figure S1. c Unstarved BMMCs were left untreated (con) or stimulated with Ag (20 ng/ml) for 20 min. Degranulation was determined by β-hexosaminidase assay. Each point is the mean of triplicates ± SD. d BMMCs were left unstimulated (-) or stimulated with Ag (200 ng/ml) for the indicated time points. Whole-cell lysates were subjected to anti-SHIP1 immunoprecipitation (IP) and WB analysis was performed with antibodies against P-Tyr (upper panel) and SHIP1 (lower panel). e BMMCs 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-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 200 ng/ml Ag (see Fig. 2e). f BMMCs were left unstimulated (con) or stimulated with Ag (20 ng/ml) for 4 h. Secreted IL-6 (left) and TNF-α (right) were measured by ELISA. Each bar is the mean of triplicates ± SD. Comparable results were obtained with cells from different BMMC cultures (n = 3 (a, b, d); n > 3 (c, e, f))