SAP blocks binding of SHP-2 to phosphorylated Ly-9 and CD84.

To detect interactions between Ly-9 or CD84 and SHP-2, COS-7 cells were cotransfected with cDNAs encoding one of the receptors, humanc-fyn, in the presence or absence of human SAP. On immunoprecipitation with monoclonal antibody directed at either Ly-9 or CD84, the receptor-associated protein complexes were analyzed by Western blotting. (A) SHP-2 binds to phosphorylated Ly-9 in the absence of SAP. COS-7 cells were transfected with the indicated constructs, and 48 hours later cell surface proteins were biotinylated, cells were lysed, and Ly-9 was immunoprecipitated as described in “Materials and methods.” Immunoprecipitates and whole-cell lysates were run on SDS-PAGE and transferred to PVDF. Ly-9 was detected using streptavidin conjugated to horseradish peroxidase, and phosphotyrosine, SAP, and SHP-2 were detected using the antibodies described in “Materials and methods.” Whole-cell lysates were Western blotted for SAP and SHP-2 to control for gel loading. (B) SHP-2 binds to phosphorylated CD84 in the absence of SAP. COS-7 cells were transfected with the indicated constructs, and 48 hours later cell surface proteins were biotinylated, cells were lysed, and CD84 immunoprecipitated as described in “Materials and methods.” Immunoprecipitates and whole-cell lysates were run on SDS-PAGE and transferred to PVDF. CD84 was detected using streptavidin conjugated to horseradish peroxidase, and phosphotyrosine, SAP, and SHP-2 were detected using the antibodies described in “Materials and methods.” Whole-cell lysates were Western blotted for SAP and SHP-2 to control for gel loading.