Fig. 7.
Validation of the SLP76-Bcr interaction in activated mast cells. A, Co-purification of Bcr with SLP76OST. Slp76OST/OST BMMC sensitized with IgE anti-DNP were challenged with DNP-HSA (+) or without (−) for 2 min at 37 °C and lysed. Samples of whole cell lysates (WCL) were Western blotted with anti-Bcr antibodies. Remaining lysates were subjected to affinity-purification with Strep-Tactin. Eluates were electrophoresed and Western blotted with anti-SLP76, anti-phosphotyrosine (pY), and anti-Bcr antibodies. B, Co-immunoprecipitation of SLP76 with Bcr. Slp76+/+ BMMC sensitized with IgE anti-DNP were challenged with DNP-HSA (+) or without (−) for 2 min at 37 °C and lysed. Samples of whole cell lysates (WCL) were Western blotted with anti-Bcr or anti-phospho-PLCγ-1 antibodies. Remaining lysates were immunoprecipitated with anti-Bcr antibody. Eluates were electrophoresed and Western blotted with anti-Bcr, or anti-SLP76 antibodies. C, Inducible Bcr phosphorylation in activated mast cells. BMMC sensitized with IgE anti-DNP were challenged with DNP-HSA (+) or without (−) for 2 min at 37 °C and lysed. Samples of whole cell lysates (WCL) were Western blotted with anti-Bcr, anti-pY178 Bcr, anti-phospho-SLP76 or anti-Grb2 antibodies. (D, E) Genetic evidence that Bcr is involved in FcεRI signaling. Aliquots of BMMC from Bcr−/− and from Bcr+/+ littermate control mice were lysed in SDS. whole cell lysates (WCL) were Western blotted with anti-Bcr antibodies and, as positive controls, with anti-Dok-3 or anti-Grb2 antibodies (D). Aliquots of the same cells were sensitized with IgE anti-DNP, and challenged with the indicated concentrations of DNP-HSA. β-hexosaminidase was measured in supernatant 10 min later (E).