Fig. 3.

Detection and quantification of the C2-sequence point mutants within the plasma membrane of transfected cells by flow cytometry with the 5D3 surface antibody. Stably-transfected HEK293 cells were incubated with phycoerythrin-labeled 5D3 antibody (blue line) or a negative control antibody (red line) (a). Similarly to wild-type ABCG2, the C2-sequence mutants and the R482G gain-of-function mutant were detected on the cell surface, and quantified by 5D3 fluorescence; the bars represent the mean of median fluorescence (b). HEK293 cells with stable expression of either pcDNA3.1 empty vector, wild-type ABCG2 or the C2-sequence L352A mutant were fixed and immunostained with the ABCG2-specific BXP-21 antibody, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (c)