Analysis of Nef functions. (A) Analysis of NF-AT-activity. A.301 cells were co-transfected with a NF-AT-Luc-reporter plasmid and expression plasmids containing PBj-nef-wt, PBj-nef-202/20GG or pGL3-Basic as control. 16 h prior lysis, cells were stimulated with TPA and ionomycin (white bars) or solvent control (black bars). Mean of results of dual luciferase assays in triplicates is shown as relative luciferase units (RLU) (**, P < 0.04 compared to control; ns, P > 0.25). (B) Binding of the Golgi adaptor complex AP-1. GST-fusion proteins containing the C-terminal part (aa109-261) of PBj-wt and PBj-Nef202/203GG Nef was used to precipitate AP-1 from lysates of non-stimulated T cells, and GST served as control. Precipitates were analyzed by Western Blot using an AP-1 γ-subunit (γ-adaptin) detecting antibody (upper panel) or GST detecting antibody (lower panel). (C) Downmodulation of cell surface receptors. Analysis of Nef mediated downmodulation of CD3, CD4, CD28 and MHC-I was done and related to GFP reporter expression in Jurkat T cells transfected with respective pCG-nef-IRES-GFP plasmids as analyzed by FACS. For quantification, the levels of specific surface molecules´ expression (red fluorescence) were determined for cells expressing a specific range of GFP (n, no; l, low; m, medium; h, high expression). The extent of downmodulation (x-fold) was calculated by dividing the MFI obtained for cells transfected with the nef-minus plasmids by the corresponding values obtained for cells transfected with plasmids coexpressing Nef and GFP (NC, no Nef; SIVmac239, Nef of SIVmac239; PBj-wt, wt Nef of PBj; PBj-mut, Nef202/203GG of PBj). One representative out of 3 experiments displayed.