FIGURE 6.

MRP-1 expression and activity are YB-1-dependent. A, Western blotting shows elevated YB-1 protein expression in whole cell lysates of EOMA versus MAE cells. B and C, Western blotting (B) was done on cytosolic and nuclear fractions of MAE and EOMA cells with densitometry (C) showing elevated levels of YB-1 protein localized to the nuclear compartment of EOMA cells. D, association of MRP-1 with YB-1 in cell nuclei was identified by co-immunoprecipitation (IP). Cell lysates (200 μg) were immunoprecipitated with YB-1 antibody or control IgG, and the immunoprecipitates were resolved with SDS-PAGE and immunoblotted (IB) for MRP-1 and YB-1. Transient transfection with YB-1 siRNA showed decreased MRP-1 activity (E) by means of a calcein clearance test and increased cell death measured by an LDH toxicity assay (F). Total (G) as well as nuclear (H) MRP-1 expression was decreased after YB-1 knockdown in EOMA cells. Results are expressed as mean ± S.D. (error bars); *, p < 0.05.