Interactions of SAP with the phosphorylated cytoplasmic tail of CD84.

(A) CD84 and SAP in yeast. The interaction of SAP with the cytoplasmic tail of human CD84 in the presence or absence of Fyn _{420, 531 Y-F} took place in the yeast cell and was measured in a β-galactosidase assay_. For each construct, at least 3 independent colonies were tested in the galactosidase assay. Open bars: cells transfected with empty pBRIDGE vector and pGAD424 encoding the cytoplasmic tail of CD84; solid bars: cells transfected with pBRIDGE-SAP and pGAD424 encoding the cytoplasmic tail of CD84; hatched bars: cells transfected with pBRIDGE-SAP and Fyn_{420, 531 Y-F} and with pGAD424 encoding the cytoplasmic tail of CD84. Control, empty pGAD424 with pBRIDGE encoding the indicated DNA sequences. (B) SAP coprecipitates with phosphorylated CD84 in a B-cell line. Cells of a variant of the Burkitt lymphoma cell line Raji, which was known to express SAP in its cytoplasm,2 were biotinylated and incubated in the presence or absence of 1 mM pervanadate. After lysis in detergent, CD84 was immunoprecipitated with 1 μg antihuman CD84 monoclonal antibody (IP α-CD84) (CD84.1.21). Proteins were transferred to PVDF membranes and Western blotted (WB) with streptavidin (WB streptavidin), antiphosphotyrosine (α-pY), and monoclonal antihuman SAP (α-SAP)10C4.2 CONTROL, immunoprecipitation with mouse immunoglobulin. (C) SAP coprecipitates with phosphorylated CD84 in Jurkat. Stable CD84 transfectants of the T- cell line Jurkat were biotinylated and then incubated in the presence or absence of 1 mM pervanadate. After lysis in detergent, CD84 was immunoprecipitated with 1 μg antihuman CD84 monoclonal antibody (IP α-CD84) (CD84.1.21). Proteins were transferred to PVDF membranes and Western blotted (WB) with streptavidin (WB streptavidin), antiphosphotyrosine (α-pY) and monoclonal antihuman SAP (α-SAP) (10C4.2). CONTROL, immunoprecipitation with mouse immunoglobulin.