Pulse-chase analysis of epitope-tagged ABCG5 and ABCG8 in transiently transfected CHO-K1 cells. Cells were transfected with empty plasmids (V), ABCG5-myc, ABCG8-HA, or ABCG5-myc and ABCG8-HA. Forty-eight hours following transfection, cells were washed with PBS, incubated in methionine- and cysteine-free medium for 2 hours, and then incubated in methionine- and cysteine-free medium supplemented with 100 μCi/ml [35S]-Met/Cys for 15 minutes. Cells were washed with PBS and incubated in serum-free medium for the indicated times. Cell lysates were prepared, and anti-myc mAb was used to immunoprecipitate ABCG5-myc in cells expressing ABCG5-myc alone or ABCG5-myc in the presence of ABCG8-HA (top panels). Anti-HA mAb was used to immunoprecipitate ABCG8 in cells expressing ABCG8-HA alone or ABCG8-HA in the presence of ABCG5-myc (bottom panels). Immunoprecipitated proteins were resolved by SDS-PAGE (4–20%) and visualized by fluorography (exposure: 8 hours, left; 36 hours, right). This experiment was repeated four times with similar results. *unidentified 48 kDa band observed in CHO-K1 cells transfected with ABCG8.