Autoprocessing of fusion precursors with different protease sequences. (A) Schematic diagram of the fusion precursors. The p6* sequence is derived from the NL4-3 strain, which contains two native cleavage sites, the distal (D) and proximal (P) sites, as indicated. (B) Space fill representation of two mature protease structures with the relevant surface amino acids highlighted in different colors. Top, an NL4-3 protease with residue S37 (the NL4-3 protease structure with N37 residue is not available); bottom, a pseudo wild type protease. (C) Autoprocessing of different precursors in transfected HEK293T cells. Post-nuclear cell lysates were prepared from cells transfected with plasmids encoding the indicated fusion precursors and subjected to western blot analysis. The blot was simultaneously probed using polyclonal rabbit anti-GST (upper) and mouse anti-HA (lower) primary antibodies, followed by IR700 goat anti-rabbit and IR800 goat anti-mouse secondary antibodies for concurrent visualization with an Odyssey infrared dual laser scanning unit. At right, the products released by the cleavage reaction at the D site are connected by a dotted line and those released by P site cleavage by a solid line.