Fig. 6

Biochemical analysis of Alix exosomal marker. After a 7 day-infection at an MOI 0.5, exosomes were purified from cell culture supernatants of infected THP-1 derived macrophages (reactivation model) and uninfected THP-1 derived-macrophages. Equal amounts (20 μg) of each sample, as well as the viral inoculum derived from the TB40E reactivation model (MOI 0.5) and a positive control (exosomes purified from COLO-1 cells), were run by SDS-PAGE and transferred to a nitrocellulose membrane, WB analysis was performed using a mouse monoclonal antibody raised against the ALIX (95 kDa) exosomal marker. The immunoreactions were revealed by AP-conjugated anti-mouse antibody. Lane 1: protein fraction obtained from the supernatant of TB40E-infected THP-1 derived-macrophages (reactivation model), as detailed in the Methods section; lane 2: protein fraction obtained from the supernatant of uninfected THP-1 derived-macrophages processed for exosome extraction, as detailed in the Methods section; lane 3: protein fraction obtained from the supernatant of infected THP-1 derived-macrophages (reactivation model) processed for exosome extraction, as detailed in the Methods section; lane 4: purified exosomes derived from the cell culture supernatant of COLO1 cells (positive control). Molecular weight markers are indicated to the left of the figure