Nef binding to AP2μ is associated with TfR upregulation. (A) 293 T cells were cotransfected with different Nef-YFP fusion protein vectors and AP2μ-CFP. 24 hours later the amount of cells scoring FRET + was measured by flow cytometry as described before . CFP + YFP is a negative control of 293 T cells which are cotransfected with CFP and YFP expressing plasmids. CFP-YFP is a positive control of 293 T cells that express a CFP-YFP fusion protein. Mean values and SD are calculated from four to nine independent transfections. (B) 293 T cells were transfected with lentiviral vectors coexpressing GFP and a shRNA against AP2μ or the GFP only expressing vector control. 72 hours later the cells were “supertransfected” with pCG-IRES-mTagBFP plasmids expressing different lentiviral Nef proteins. After additional 24 hours cells were stained with TfR-APC antibody and cell surface levels in shRNA/Nef expressing cells were assessed by flow cytometry. Mean values and SD from triplicate transfections of one representative experiment out of three is shown.