Immunoblotting analyses of ABCG5 and ABCG8 in human liver. Panel A shows the immunodetection of ABCG5 (tracks 1–6), ABCG8 (tracks 7–12) in membrane preparations from human liver (HL, tracks 1, 2, 7, 8) mouse liver (ML, tracks 3, 4, 9, 10) or rat liver (RL, tracks 5, 6, 11, 12). The anti-ABCG5 peptide antibody detected a faint mouse band, but no other specific binding was identified. Although the pre-immune sera detected bands in the rodent tissue samples, none were detected in human liver (tracks 13–16, MB, mouse brain). Specificity was further shown by pre-incubation of the antibodies with the peptides they were raised against (panel B). In the presence of the specific peptides, the 75 kDa bands are not detected in human liver microsomes. Panel C shows the results of deglycosylation of human total liver microsomes, probed with anti-ABCG5 (left hand panel), anti-ABCG8 (middle panel) or anti-transferrin (right hand panel). Aliquots from the same incubation were separated for all three western blots. Although ABCG5 and ABCG8 do not appear to have their SDS-PAGE mobility's altered by either EndoH or PNGase F treatment, that of transferrin in the same samples is clearly effected (see Text for discussion). Newly synthesized (sensitivity to EndoH), as well as mature forms of transferrin (resistant to EndoH, but fully sensitive to PNGase F) are present in the liver membrane preparations.