Known protease inhibitors block protease autoprocessing. A. HEK293T cells transfected with the indicated pEBG construct were incubated with or without protease inhibitors at increasing concentrations. Darunavir: 0.1 μM, 1 μM and 10 μM; Indinavir: 1 μM, 10 μM and 100 μM. Post-nuclear cell lysates were prepared at 40 h post-transfection and aliquots (~20 μL) of each sample were analyzed in parallel using monoclonal mouse anti-Flag, anti-HA, anti-GAPDH primary antibodies and IR800 goat anti-mouse secondary antibody. Schematic diagrams of the full length fusion precursor and processing products are indicated at left. Molecular mass markers (kDa) are indicated at right. B. HEK293T cells that were transfected with NL4-3-derived proviruses encoding the indicated proteases were incubated with or without protease inhibitors at the same concentrations as in panel A. Post-nuclear cell lysates (Cell) and VLP particles (VLP) were prepared as described (Materials and Methods) and subjected to western blot analysis using monoclonal mouse anti-p24. The full length Gag polyprotein (p55) and p24/p25 doublet are indicated at left.