Relative levels and distribution of expression of ABCG5-myc and ABCG8-HA in CRL-1601 stably transfected with empty plasmids (V), ABCG5-myc (5), or ABCG5-myc and ABCG8-HA (5+8). (a) Steady-state levels of immunoreactive ABCG5 and ABCG8 in CRL-1601 cells. Cell lysates (5 μg) were subjected to SDS-PAGE and immunoblot analysis for expression of ABCG5 (top) or ABCG8 (middle). Immunoreactive ABCG5 and ABCG8 were detected by enhanced chemiluminescence. (b) Fractionation of CRL-1601 cells that stably express ABCG5-myc alone or ABCG5-myc and ABCG8-HA. Cells were grown to 90% confluence in 10-cm dishes. Postnuclear supernatants were prepared and fractionated as described in Methods. Fractions (20%) were subjected to SDS-PAGE and immunoblot analysis for ABCG5-myc and ABCG8-HA. Separation of ER membranes from other organelles was confirmed by immunoblot analysis of marker proteins: calnexin (ER), LRP (plasma membrane and endosomes), p58K (Golgi complex), Rab11 (endosomes).