Nef expression analysis represented as variability chart for three-factors (Construct-Plant-Leaf position crossed data). The effect of different viral suppressors of gene silencing and influence of leaf position on Nef expression yield was evaluated by ELISA using a mouse monoclonal antibody against HIV-1 Nef. For this study three independent plants were agroinfiltrated with Nef, Nef/AMCV-P19, Nef/PVX-P25 or Nef/TBSV-P19 and leaf samples were collected at day 9 post infiltration from different positions on the plant (T: top leaf; M: middle leaf; B: bottom leaf). Leaf extracts (50 and 25 μg of total soluble protein (TSP)) were loaded on the ELISA plate wells as four indipendent replicas. All data were subjected to analysis of variance (ANOVA) using JMP8 Statistical Discovery software. The variability chart reports the maximum and minimum bars to show the range of measurements. Nef yield is expressed as percentage of total soluble protein (% TSP). The group means are represented by orange (Construct) and green (Plant) lines as reported in the legend. Mean values of Nef yield (% TSP) for each construct group are reported on the chart over the orange line.