HEK-293 cells stably expressing wild-type Nrf13×Flag were treated with MG132 and/or cotransin, an inhibitor of protein insertion into the Sec61 translocation channel (Garrison et al., 2005) as indicated (lanes 1 to 4) and total cell lysates were prepared. Lanes 5 and 6 contain full-length Nrf13×Flag and Nrf1(104-742)3×Flag that were translated in vitro (IVT) in rabbit reticulocyte lysate in the absence of membranes. The HEK293 cell lysates and IVT reactions were examined by SDS-PAGE followed by immunoblotting with anti-Flag antibody. Different Nrf1 species are shown (ungly–unglycosylated; unmod–unmodified). Nrf1 p120 (species ‘a’) was converted to species ‘c’, which comigrated with the primary translation product (species ‘d’) upon expression in cells treated with cotransin, suggesting that the slow mobility of p120 arose from modifications (e.g., N-linked glycosylation) that occurred within the endoplasmic reticulum. Retrotranslocation and processing of p120 (species ‘a’) yielded p110 (species ‘b’). Species ‘b’ was not sensitive to endoglycosidase H (Figure 4E), suggesting that it was deglycosylated upon retrotranslocation into the cytosol. Nevertheless, species ‘b’ migrated considerably more slowly than the primary translation product for Nrf1(104-742)3×Flag (species ‘e’), indicating that p110 must carry additional modifications that remain uncharacterized. Please note that deglycosylation by cytosolic enzymes converts the Asn at the site of glycosylation to Asp, which could influence migration on SDS-PAGE.