CD4/PIR-A3 is expressed on 293T cells and is recognized by anti-human CD4. (A) Expression of CD4/PIR-A3 in 293T cells. 293T cells were transfected with 0.5 μg of either pcDNA3 (thin trace) or CD4/PIR-A3 (thick trace) cDNA. Twenty-four hours after transfection, the cells were labeled with PE-conjugated anti-human CD4 for flow cytometric analysis. (B) Anti-CD4 immunoprecipitates CD4/PIR-A3. Twenty-four hours after transfection, 293T cells were surface labeled with biotin followed by pervandate stimulation, as indicated. The cells were lysed in a buffer containing 1% TTX-100, and lysates were immunoprecipitated with anti-CD4. Immune complexes were eluted in reducing Laemmli sample buffer, separated by 8% to 16% SDS-PAGE, and detected with SA-HRP and ECL.