Ro5-4864 affects Ag-triggered tyrosine phosphorylation events in wild-type and SHIP1-deficient BMMCs. (A) IgE-loaded wild-type and SHIP1-deficient BMMCs were pretreated for 20 min with 100 μM Ro5-4864 or the respective amount of DMSO and stimulated with Ag (DNP-HSA, 20 ng/ml) for 5 min or left unstimulated. Subsequently, cellular lysates were analyzed by immunoblotting using antibodies against phosphotyrosine (upper panel) and p85 (lower panel, loading control). (B) Wild-type BMMCs were treated as under (A) and cellular lysates were subjected to pull-down reactions with GST or GST-SH2(Lyn) proteins. Precipitated proteins were analyzed by immunoblotting against phosphotyrosine (top panel), Syk (middle panel), and FcεRIβ-chain (bottom panel). Usage of equal amounts of GST proteins was verified by Coomassie staining (data not shown). Comparable results were obtained with cells from different BMMC cultures.