Figure 2.

Determination of the expression and localization of ABCG2 variants in cellular systems in vitro. (Panel a) Expression of ABCG2 variants in HEK293 cells, detected by immunoblotting. Left Panel – immunoblot, Right Panel - expression levels of the wild type ABCG2, the ABCG2-M71V and the ABCG2-Q141K variants, normalized to beta-actin and presented as relative expression, compared to the ABCG2-Wt. Bars represents the mean relative expression (±SEM) from triplicate Western blots (Student’s t-test, *p < 0.05). The different proteins examined in the Western blot were developed by the respective antibodies and cropped from the indicated parts of the same gel and blot. (Panel b) Cell surface membrane expression of ABCG2 variants in HEK293 and HeLa cells, detected by the 5D3 antibody reacting with an extracellular epitope. Flow cytometry measurements (±SEM; Student’s t-test, *p < 0.05, **p < 0.01, compared to the expression of the wild type ABCG2 in the same cell type). (Panel c) ABCG2 and ABCG2-M71V protein expression visualized by confocal microscopy in transfected HEK293 cells. ABCG2 is labeled with BXP-21 antibody (red), and nuclei are stained with DAPI (blue). Scale bars represent 20 µm. (Panel d) Localization of ABCG2 and ABCG2-M71V protein as observed by confocal microscopy in transfected and polarized MDCKII cells. ABCG2 is stained with the BXP-21 antibody (green), and the basolateral membrane marker Na⁺/K⁺ ATPase is stained with a specific antibody (red). Yellow arrows point to the apical membrane.