CD4/PIR-A3 is expressed and associates with FcɛRIγ in ANA-1 stable transfectants. (A) ANA-1 express CD4/PIR-A3 and CD4/PIR-A3^R632L. ANA-1 macrophages were transfected with pcDNA3 (thin traces), CD4/PIR-A3 (left panel, thick trace), or CD4/PIR-A3^R632L (right panel, thick trace) expression vectors, as described in Materials and Methods. Clones were screened for expression using PE-conjugated anti-human CD4. (B) CD4/PIR-A3 is associated with a tyrosine phosphoprotein after pervanadate stimulation in ANA-1. ANA-1 clones (5 × 10⁶ cells/point) were stimulated in the absence or presence of pervanadate followed by lysis in 1% Brij-96 buffer. Whole cell lysates were immunoprecipitated with anti-human CD4 and eluted in nonreducing Laemmli sample buffer. Immune complexes were separated by 4% to 20% SDS-PAGE and immunoblotted with antiphosphotyrosine. (C) FcɛRIγ associates with CD4/PIR-A3 via Arg⁶³². ANA-1 clones (25 × 10⁶ cells/point) were lysed and processed as above, using reducing Laemmli sample buffer followed by separation with 4% to 20% SDS-PAGE and immunoblotting with antimurine FcɛRIγ.