Coprecipitation of ABCG5 and ABCG8 in transiently transfected CHO-K1 cells. (a) Cells were transfected with empty plasmid (V), ABCG5-myc (5), ABCG8-HA (8), or ABCG5-myc plus ABCG8-HA (5+8). Forty-eight hours following transfection, cell lysates were prepared and aliquoted into two tubes. ABCG5-myc and ABCG8-HA were immunoprecipitated with mAb’s directed against the myc (left) and HA epitopes (right), respectively. Duplicate aliquots (10%) of each immunoprecipitate (P) and supernatant (S) were subjected to SDS-PAGE (8%) and transferred to nitrocellulose membranes. Each membrane was subjected to immunoblot analysis using pAb’s directed against the myc (G5, top) or HA (G8, bottom) epitopes. (b) Cells were transfected with ABCG5-myc, ABCG5-HA, ABCG8-myc, or ABCG8-HA (left). Cell lysates from a 60-mm dish of each of the following transfections were pooled (left): empty plasmids (V), ABCG5-myc and ABCG5-HA (5&5), ABCG5-myc and ABCG8-HA (5&8), and ABCG8-myc and ABCG8-HA (8&8). Two dishes were cotransfected with each of the following construct pairs (right): ABCG5-myc and ABCG5-HA (5+5), ABCG5-myc and ABCG8-HA (5+8), and ABCG8-myc and ABCG8-HA (8+8). Lysates were aliquoted into two tubes and immunoprecipitated with mAb’s to the myc (bottom) or HA epitopes (top). Duplicate aliquots of each immunoprecipitate and supernatant (10%) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Each membrane was subjected to immunoblot analysis using pAb’s against the myc (top) or HA (bottom) epitopes. IP, immunoprecipitating; IB immunoblotting.