Western blot analysis of the post nuclear supernatant (PNS) and enriched lysosomal (Lys) fractions separated magnetically from extracts of N-acetyltransferase-His8Flag permanently transfected HeLa cells loaded with iron-dextran (FeDex).

After SDS-PAGE the proteins in the PNS and Lys fractions (1 µg total protein from each fraction was loaded) were visualized with the N-terminal N-acetyltransferase antibody (N-term). The nonspecific ∼50 kDa band visualized with the N-terminal antibody is marked as “X”. As control for the enrichment of lysosomes in the “Lys” fraction, the blot was stripped and re-probed with an antibody against human lysosomal ß-hexosaminidase A (Hex A). Furthermore, markers for the ER (Calnexin) and early endosome (EEA1) were visualized after stripping and re-probing the blot with the appropriate antibody (bottom).