Fig. 2

HIV Nef activates the human c-MYC promoter, but not the AICDA promoter. a. Bar graphs showing promoter activities of c-MYC (top panel) and AICDA (bottom panel) when exposed to Nef, relative to control (empty vector). In both cases, the promoter-luciferase reporter construct (500 ng per well in 35 mm dish) was co-transfected into HT1080 cells with the internal control pRL-TK (50 ng) in the presence of increasing concentrations of the Nef expressing vector pcDNA-Nef (0–500 ng) or empty vector (pcDNA3.1). Luciferase activity was measured 30 h post-transfection and normalized to renilla luciferase activity. Activation fold values were calculated by setting promoter activity exposed to pcDNA3.1 empty (500 ng) to 1. Mean values (±) were calculated from three independent experiments. Bars indicate standard deviation. *** p ≤ 0.001; **p ≤ 0.01; * p ≤ 0.05. All experiments were performed in triplicate. b. To demonstrate that increasing amounts of Nef expressing plasmid leads to increasing expression of Nef protein, western blot analysis of whole proteins lysates from luciferase assays performed in HT1080 cells above was performed. Proteins were separated on 15% SDS-PAGE, transferred onto nitrocellulose membranes and analysed using an antibody specific to Nef. p38 was detected as a loading control to demonstrate equal loading of protein lysates