Time course expression analysis of Nef in vacuum agroinfiltrated N. benthamiana plants. Six weeks old Nicotiana benthamiana plants were vacuum-infiltrated with Agrobacterium. tumefaciens clones harbouring Nef, Nef/AMCV-P19, Nef/TBSV-P19 and Nef/PVX-P25. Leaf samples from three different plants were collected on 3, 5, 7, and 9 days post infiltration (d.p.i.). Leaf extracts were normalised for total soluble protein content (TSP) and analysed by direct ELISA using a mouse monoclonal antibody to HIV-1 Nef. For all samples a total of 50 μg of TSP were loaded in each ELISA plate well. C+: 100 ng of bacterially-derived Nef; C-: leaf extract of plants infiltrated with infiltration buffer. Values are the mean ± standard error of the mean (SEM) of triplicate samples.