Analysis of Nef HLA-I downregulation capacity for LANL 2004 consensus B and C Nef sequences. Consensus B and C Nef sequences were obtained from the Los Alamos National Laboratory HIV Sequence database (2004 update), synthesized and tested. Panels A and B : Representative flow cytometry plots depict expression of surface HLA-A*02 (y-axis) and green fluorescent protein (GFP, x-axis), a marker of Nef-transfected cells. Representative flow cytometry plots for cells transfected with consensus B Nef plasmid (A) or consensus C Nef plasmid (B) are shown. HLA-I downregulation activity was assessed in tertiles of cells expressing low, medium (med), or high amounts of Nef, as indicated by the level of GFP co-expressed by the dual promoter plasmid. Panel C: Results from ten replicate assays are shown, indicating a reduced ability of consensus C Nef to downregulate HLA-I, particularly in cells expressing the lowest amount of Nef protein. For comparison, the outcome when all cells are gated together is also presented. Bars and whiskers represent the mean and standard error of the mean for each group, respectively.