HTS Assay for Functional Modulators of ABCB6 Figure 5. Selectivity and validation of HTS identified compounds by hemin agarose affinity chromatography. (a) verteporfin and (b) tomatine hydrochloride potently disrupt the interaction between ABCB6 and hemin-agarose compared with (c) succinylacetone. (d, e and f) image J analysis of ABCB6 band intensity treated with (d) verteporfin, (e) tomatine hydrochloride and (f) succinylacetone averaged over three independent experiments. Mitochondria isolated from K562 cells expressing ABCB6-Flag or the empty vector were incubated in the presence or absence of increasing concentration of the indicated compound and hemin-agarose and the resulting complex was immunoblotted using a monoclonal antibody to the flag-tag. Results are representative of 3 independent experiments. ‘*’ significantly different from untreated controls. P,0.05. ‘NS’ differences are non-significant compared to untreated control. doi:10.1371/journal.pone.0040005.g005 transport of hemin into isolated mitochondria, despite the fact that it does not interfere with hemin binding to ABCB6 suggests that benzethonium chloride may function as a potential inhibitor of ABCB6 by interacting with a site that is different from that bound by hemin. These results presented above highlight the recurring theme in the transporter field where the transporters appear to have multiple substrate binding sites that promote co-operativity either in promoting or inhibiting transport of a specific substrate. In contrast to benzethonium chloride, both tomatine hydrochlo- ride and verteporfin were capable of inhibiting transport as well as PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e40005