Figure 3.

Passive activation of FcγRIIB depends on Lyn as well as SHIP1 and contributes to SHIP1 membrane recruitment. (A) Murine WT, Lyn^−/− and Lyn/ Ship1^−/− BMMCs sensitized with SPE-7 (0.15 μg/ml), were challenged as indicated for 2 min. Cells were lysed and subjected to SDS-PAGE and subsequently to P-FcγRIIB and FcγRIIB-specific Western blotting (loading control: anti-p85). Pictures of the full blots are shown in Supplementary Figure 16. (B) WT and Ship1^−/− BMMCs sensitized with SPE-7 (0.15 μg/ml) were stimulated as indicated. Cells were lysed and subjected to SDS-PAGE and Western blotting. Pictures of the full blots are shown in Supplementary Figure 17. (C) WT and Ship1^−/− BMMCs were sensitized with SPE-7 (0.15 μg/ml). Cells were treated with 2 mM of the phosphatase inhibitor PV or the respective amount of the solvent H₂O for 1 h prior to 2 min of Ag stimulation with the depicted concentrations of DNP-HSA. Cells were lysed and subjected to SDS-PAGE and Western blotting. Pictures of the full blots are shown in Supplementary Figure 18. (A–C) As proteins of comparable size were analyzed, lysates were separated on two gels, which were blotted on two membranes with an anti-p85 loading control on each membrane (detections belonging to one membrane marked with/without asterisk). (D) SPE-7-sensitized (0.15 μg/ml) WT and Fcgr2^−/− BMMCs were challenged as indicated. MCs were lysed and immunoprecipitation against SHIP1 was conducted. SHIP1 tyrosine phosphorylation was significantly decreased in Fcgr2^−/− BMMCs (p = 0.0252). The bar graph shows the relative phosphorylation of SHIP1 normalized to the level of SHIP1 phosphorylation after 2,000 ng/ml DNP-HSA in WT BMMCs. Pictures of the full blots are shown in Supplementary Figure 19. Results shown in this figure are representatives of at least 3 independent experiments and in the diagram depicted as mean ± SD.