Dynabeads 280 streptavidin

To collect viral particles, culture supernatant harvested at 48 h post-transfection of viral constructs was centrifuged at 1,000 ×g for 10 min and then filtered through a 0.45 μm filter to remove cell debris and large aggregates. Subsequently, 20 μL of Dynabeads®280 streptavidin (Life Technologies), precleaned twice with 1× PBS, was added into every 10 mL of the supernatant in order to assist the visualization of pellet after ultracentrifugation (100,000 ×g for 1 h). The viral particles and the beads were resuspended in lysis buffer (0.5% Triton X-100, 50 mmol/L pH = 7.5 Tris-HCl, 300 mmol/L NaCl) containing protease inhibitor cocktail (1:100). The lysates and the beads were further separated by microcentrifugation. To collect cell lysates after removing culture supernatants, the cells were washed once in cold 1× PBS, trypsinized, pelleted, and finally lysed in lysis buffer containing protease inhibitor cocktail (1:100). Gag from both supernatant and the cell lysates were analyzed by SDS-PAGE on 10% acrylamide gels and transferred to Immobilon^TM-P membranes (Millipore). Immunoblotting was carried out with HIV-Ig or anti-GFP antibodies. Release efficiency was calculated (at 48 h post-transfection) as the ratio of supernatant Gag to total Gag, both determined by densitometry analysis of Western blot images using Fiji software.