A3G and A3F concentrations are similarly distributed between cells and VLPs by RNA. (A)

<p>293T cells were co-transfected at a 1:5 ratio with expression vectors for T7-tagged A3G or A3F, and Wt Gag. Cell lysates (CL) were kept for analysis. Supernatant from cells transfected with an empty plasmid (negative control) and VLPs were harvested and isolated by ultracentrifugation through a continuous sucrose gradient (20–60%). Fractions were harvested and p24^Gag-containing fractions were identified by immunoblot. T7-tagged A3G was purified and quantified as specified in the material and methods. Cell lysates, fractions containing VLPs (or the corresponding fraction from the negative control) and purified A3G were analysed by immunoblot. Gag and HSP90 were visualised using anti-p24^Gag and anti-HSP90 antibodies, respectively. A3G and A3F were visualised using an anti-T7 antibody. APOBEC3 proteins were quantified by densiometry. This figure shows a representative example. <b>(B)</b> Standard curve of purified T7-A3G visualised and quantified by densiometry of the immunoblot of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004609#ppat.1004609.g007" target="_blank">Fig. 7A</a>. <b>(C)</b> RNA was extracted from cell lysates (CL) and VLP samples and quantified. Total A3G/F protein in CL and VLPs was quantified using a standard curve. The RNA in the negative control was below the detection threshold of the method. <b>(D)</b> The total amount of A3G or A3F was divided by the total concentration of extracted RNA. The graph shows the average of 4 independent experiments. Error bars indicate standard deviation. There was no statistically significant difference between the ratios in lysates and VLPs (p>0.5).