Identification of phosphotyrosine-dependent and -independent SAP binding motifs in CD150.

(A) COS-7 cells transiently transfected with CD150, or CD150 constructs with SAP motif tyrosine mutations (Figure 2 lists nomenclature) plus SAP-FLAG with or without Fyn coexpression. CD150 immunoprecipitation followed by antiphosphotyrosine, streptavidin-HRP, and anti-FLAG Western blot. (B) Yeast 2-hybrid analysis of SAP binding to non–tyrosine-phosphorylated CD150 or CD150 with tyrosine-phenylalanine mutations. ▪, +SAP; ■, +pBridge. Quantitation of β-galactosidase activity was performed as described in “Materials and methods.”