Temporal analysis of protease autoprocessing. pEBG-derived plasmids expressing the indicated fusion precursors were transfected into HEK293T cells using calcium phosphate. Post-nuclear cell lysates were prepared at the indicated times post transfection and subjected to western blot analysis using polyclonal rabbit anti-GST (top) and mouse anti-HA (middle) primary antibodies, and IR800 goat anti-rabbit and IR700 goat anti-mouse secondary antibodies. The same blot was stripped and analyzed using mouse anti-GAPDH (bottom) as a loading control. The full length fusion precursor and processing products are indicated at left; molecular mass markers (kDa) are indicated at right.