Figure 5.

Effects of mutations of Asn316 and Phe320 on ABCG1 trafficking. Panel A: Biotinylation of cell surface proteins. HEK293 cells transiently expressing WT or mutant ABCG1 were biotinylated exactly as described in Materials and Methods. Biotinylated cell surface proteins (biotinylation) and total proteins from whole cell lysate were analyzed by 8% SDS-PAGE and immunoblotting. ABCG1 and calnexin were detected using a polyclonal anti-ABCG1 antibody, H-65, and a monoclonal anticalnexin, respectively. Similar results were obtained from at least two more independent experiments. Panel B: Immunofluorescence of wild type and mutant ABCG1. The subcellular localization of wild type and mutant ABCG1 in stably or transiently transfected HEK 293 cells was determined by confocal microscopy as described in the legend to Figure 4A. ABCG1 was detected with H-65 and indicated in green. Nuclei were stained with DAPI and shown in blue. The plasma membrane marker, Na⁺K⁺-ATPase was shown in red. Transfectants tested expressed either wild type or mutant ABCG1 as indicated in the figure (magnification: 100× ).