Autoprocessing of GST- miniprecursor fusions in E. coli and HEK 293T cells. A. Bacteria E. coli BL21(DE3) bearing pGEX-3X derived plasmids encoding the indicated miniprecursor construct were induced with 40 μM of IPTG to express fusion proteins. Total cell lysates were subjected to 12% SDS-PAGE and western blotting using monoclonal mouse anti-Flag and polyclonal rabbit anti-PR primary antibodies and IR700 goat anti-mouse and IR800 goat anti-rabbit secondary antibodies. Images of both channels are presented. Samples were run on the same gel but lanes were re-arranged for presentation. Schematic diagrams of the full-length fusion precursor and processing products are indicated at left. B. HEK293T cells were transfected with pEBG-derived plasmids expressing the indicated fusion protein using the calcium phosphate method. Post-nuclear cell lysates were prepared at 40 h post-transfection and analyzed by 12% SDS-PAGE and western blotting. Aliquots (~ 20 μL) of each sample were examined in parallel with either monoclonal mouse anti-Flag or anti-HA primary antibody and IR800 goat anti-mouse secondary antibody. Molecular mass markers (kDa) are indicated at right.