Endoglycosidase treatment of recombinant, epitope-tagged ABCG5 and ABCG8 in CHO-K1 cells. Cells were transfected with empty plasmid (V), ABCG5-myc, ABCG8-HA, or ABCG5-myc plus ABCG8-HA. Cell lysates were prepared after 48 hours and aliquoted into two tubes. ABCG5-myc and ABCG8-HA were immunoprecipitated using mAb’s directed against the myc (top) and HA epitopes (bottom), respectively. Protein A-agarose beads were removed, and the immunoprecipitated proteins were incubated in the presence of PNGase F Endo H, neuraminidase, and neuraminidase followed by O-glycosidase. Proteins were precipitated with 0.015% deoxycholate and 1% trichloroacetic acid solubilized in protein sample buffer, and subjected to SDS-PAGE (8%). Proteins were transferred to nitrocellulose membranes and subjected to immunoblot analysis using rabbit IgGs directed against the myc (ABCG5) and HA (ABCG8) epitopes.