Figure 4.

Specific [¹²⁵I]IAAP photoaffinity labeling of crude membranes derived from HeLa cells expressing wild-type ABCG2 (R482wt) and the ABCG2 variants R482G, R482H, R482K, R482P, R482T, and R482Y. Crude membrane extracts (140 μg) derived from HeLa cells were labeled with [¹²⁵I]IAAP alone (−) or in the presence of 10 μM prazosin (+) as described in Materials and Methods. After labeling at room temperature for 10 min and UV cross-linking (365 nm) for 20 min on ice, the samples were subjected to immunoprecipitation using 4 μg BXP-21 antibody. The samples were then separated by SDS-PAGE (7.5%), and the gels were dried and exposed to autoradiography film for 4–7 d at −80°C. The positions of ABCG2 are denoted by brackets.