Intracellular localization of WT HFE and C282Y HFE in HEK293 transfected cells. (A) confocal microscopy of cells cultured for 24 h after transfection with EV-GFP, WT-HFE GFP and C282Y-HFE GFP stained with an antibody for the ER-resident protein calnexin and probed with Texas Red labeled secondary antibody. In the merged images, the yellow color corresponds to areas in which the green and red stainings overlap. The nucleus appears blue due to DNA staining with 4',6-diamidino-2-phenylindole. Scale bar, 10 μm. (B) HFE expression in transfected HEK293 cell lysates. HEK 293 cells (6 × 106) were transfected with pcDNA3.1 (empty vector, EV), WT HFE, or C282Y HFE (M) expression plasmids. C282Y HFE-His tag protein production in lysates was measured by ELISA. C282Y HFE-His tag-transfected cells showed markedly accelerated degradation of the C282Y HFE protein compared to N HFE protein (**, p = 0.0012) at 24 hours compared to WT HFE expression. Assays were performed in triplicate.