PxxP domain of Nef is required for the FasL induction. (A) Loss of the PxxP domain of Nef significantly diminishes the phosphorylation of p38. Western blot analysis of extracts derived from Jurkat or U937 cells transfected with either the expression vector for wild-type Nef or the amino acid-mutated Nef. Cell extracts were prepared 48 hours after transfection as described in “Materials and methods” and subjected to 12% SDS-PAGE followed by PVDF membrane transfer and analyzed by Western blotting using specific p38 and phospho-p38 antibodies as indicated. Note, the amino acid-mutated Nef (pNef^(PxxP)) failed to induce phosphorylation of p38. (B) Comparison of FasL induction by wild-type Nef versus amino acid-mutated Nef. Jurkat T cells or U937 cells were electroporated with 5 μg wild-type Nef, amino acid-mutated Nef, or wild-type Nef plus p38 inhibitor (1 μM). At 48 hours after transfection, the surface levels of FasL were determined by flow cytometry by staining with a FasL-specific antibody. Transfection efficiency was monitored by cotransfection of a pCMV plasmid encoding GFP. Thick line histograms show the indicated surface markers, and filled histograms represent the isotype-matched control antibodies. Similar results were obtained in 3 independent experiments. (C-D) The PxxP domain of Nef (amino-acid mutated) is essential for induction of FasL promoter activity in T cell or monocytic cells. Jurkat T cells or U937 cells were transfected with 5 μg hFasL-Luc promoter plasmid, wild-type Nef plus hFasL-Luc promoter plasmid, or amino acid-mutated Nef plus hFasL-Luc promoter plasmid as indicated. Luciferase activity in whole-cell lysates was assayed after 12 to 18 hours and is shown as the mean value ± SEM. Transfection efficiency was monitored by cotransfection of a plasmid encoding β-gal and results were normalized to β-gal levels. Similar results were obtained in 3 independent experiments.