PK11195 apparently binds Pgp at PM sites distinct from CSA-binding sites. (A) Mitochondrial (MITO) and PM fractions were prepared from DOX40 MM cells by high-speed centrifugation. Fraction purities were confirmed in Western blot assays using antibodies recognizing mitochondrial-specific proteins (VDAC, COXIII), and the C219 antibody was used to recognize Pgp in PM fractions. ³H-PK11195 and ³H-CSA binding was measured in PM fractions and competed by PK11195 and CSA, respectively, but PK11195 and CSA did not cross-compete in these assays, further demonstrating that PK11195 binding is CSA independent. (B) PK11195 (▪) and CSA (▨) increased Rhodamine (RHO), Hoechst 33342 (Hoechst), and BODIPY-prazosin (PRAZ) retention in DOX40 cells, suggesting that both modulate multiple Pgp substrate-binding sites. PK11195 blocked RHO efflux significantly more than CSA, suggesting that PK11195 binds particular Pgp-binding sites more efficiently than CSA. Efflux data from at least 3 assays are presented, as in Figure 1.