Fig. 5. Two functional NBDs are necessary for proper ATP hydrolysis. (A) Membrane fractions from stably co-transfected HEK293 cells. The blots are probed with BXP-21 (total ABCG2), MYC (cmyc) or HA antibodies. In all blots an anti-ß-actin antibody was included as a loading control. (B) Relative expression of HA-tagged and MYC-tagged ABCG2 in co-transfected cells. The expression was assessed by densitometry analysis of western blots of the co-transfected cells blotted with either an anti-MYC or an anti-HA antibody. The histograms are mean±s.e.m. expression normalized to ABCG2-wt-MYC (n=3). (C) ATPase activity measurements on membrane fractions analyzed. Phosphate release was measured in response to increasing concentrations of prazosin. Data are mean±s.e.m. of four experiments performed in triplicate. The EC₅₀ (µM) and V_max (nmol phosphate/minute/mg protein) were determined by non-linear regression analysis using Sigmaplot.