Figure 4From: High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle VirusCorrelation between P19 protein accumulation and levels of Nef specific siRNAs. (A) ELISA of leaf extracts from Nicotiana benthamiana plants agroinfiltrated with Nef, Nef/TBSV-P19 and Nef/AMCV-P19 was performed using a rabbit polyclonal antibody against TBSV-P19. Leaves were collected at 9 d.p.i. and plant extracts were normalized for total soluble protein (TSP). Fifty micrograms of TSP were loaded in each ELISA plate well. Values are means of triplicate determinations ± standard errors of the means. C-: leaf extract of plants infiltrated with infiltration buffer. (B) Detection of Nef specific siRNAs. Total RNA (15 μg) extracted from leaves agroinfiltrated with Nef, Nef/AMCV-P19, Nef/TBSV-P19, and from mock infiltrated plant used as a control (WT), was separated on denaturing 15% (w/v) polyacrylamide gel with 8 M Urea, stained with ethidium bromide to display relative amounts of rRNA and transferred to a positively charged nylon membrane. Nef specific digoxigenin-labelled RNA (+) (618 nt) was used as probe. M: si RNA low molecular weight RNA marker (synthetic siRNA duplexes 17, 21 and 25 bp).Back to article page