Detection of HIV-1 and Human Proteins in Urinary Extracellular Vesicles from HIV+ Patients

Figure 1

Detection of HIV-1 proteins by western blot. Extracellular vesicles were isolated from four ml of urine from HIV-1+ patients and HIV-1 negative individuals by Amicon ultrafiltration (MW cutoff = 100,000 kD). The western blot is representative of 9 HIV+ and 3 HIV-negative samples (c1, c2, and c3). Recombinant HIV Nef and p24 were added as positive controls (last panels on the right). Samples were isolated in a 4–20% gradient SDS gel and transferred to a PVDF membrane. The filter was incubated with the primary antibody, pooled HIV-1 positive plasma (bottom panels), or a monoclonal anti-HIV Nef (top panels). The secondary antibody, goat anti-mouse IgG for the anti-Nef blots or rabbit anti-human IgG for the anti-HIV antibodies, conjugated to horseradish peroxidase. Super Signal West Femto was used as chemiluminescent substrate for detection.