Figure 5.

Detection of Nef^mut/E7 based fusion products in transfected cells and exosomes. Shown is the Western blot analysis of total lysates from the same number of HEK293T cells transfected with DNA vectors expressing either Nef^mut/E7 or GC-AG Nef^mut/E7 (left panels). Equal volumes of buffer, where purified exosomes were resuspended after differential centrifugations of the respective supernatants, were also analyzed (right panel). As control, conditions from mock-transfected cells, as well as cells transfected with Nef^mut alone, were included. Polyclonal anti-Nef Abs served to detect Nef^mut-based products, while β-actin and Alix were revealed as markers for cell lysates and exosomes, respectively. Concerning signals from cell lysates, intensity ratios compared to the Nef^mut control condition, considering the respective β-actin signals, were 1.05 and 0.91 for GC-AG Nef^mut/E7 and Nef^mut/E7, respectively. Regarding exosomes, the intensity ratio between Nef^mut and Nef^mut/E7 conditions, considering the respective Alix signals, was 0.81. Molecular markers are given in kDa. The results are representative of five independent experiments. Uncropped blots showing all the bands with all molecular weight markers are shown in Supplementary Figure S3.