Figure 2. Transport of ¹⁴C-DOX, ³H-DNPSG, ³H-sunitinib and ³H-sorafenib by reconstituted RLIP76 purified from Caki-2 cells.

DNP-SG affinity purified RLIP76 from Caki-2 kidney cancer cells was applied to SDS-PAGE and subjected to Western blot analyses against anti-RLIP76 IgG as a primary antibody. SDS-PAGE was stained with Coomassie Brilliant Blue-R250 and Western blots were developed using horseradish peroxidase-conjugated goat-anti-rabbit-IgG as secondary antibody (panel A). Purified RLIP76 was reconstituted into artificial liposomes and uptake of radio-labeled ¹⁴C-DOX, ³H-DNPSG, ³H-sunitinib and ³H-sorafenib by control- or RLIP76-proteoliposomes were measured in the presence or absence of ATP. Vesicles equivalent to 250 ng of purified RLIP76 were used per 30 μl filtered reaction mixture in all panels except C. Total radioactivity retained by 0.45 μm filters in 96-well plates was determined after solubilizing filters in scintillation fluid, converted to pmol using the specific activity of ¹⁴C-DOX (87.4 cpm/pmol), ³H-DNPSG (3.7 cpm/pmol), ³H-sunitinib (43.5 cpm/pmol) and ³H-sorafenib (39.2 cpm / pmol), Each point represents an average and SD calculated from 12 measurements [control (−P) or RLIP76-proteoliposomes (+P), without (−A) or with ATP (+), in triplicates]. Uptake in RLIP76-proteoliposomes with ATP is significantly greater than other groups (p<0.005) (panel B). All transport studies were carried out using 250 ng protein/assay except when protein was varied (panel C). Incubation time was 5 min except for time dependence studies (panel D). Temperature was 37 °C except for temperature dependence studies (panel E). External sucrose concentration was 40 mM, except for studies of osmolar dependence (panel F). ATP was 4 mM except in ATP-dependence studies (panel G). DOX was 3.6 μM except in DOX-dependence studies (panel H). DNP-SG was 100 μM except in DNPSG-dependence studies (panel I). Sunitinib was 10 μM except in sunitinib-dependence studies (panel J). Sorafenib was 10 μM except in sorafenib-dependence studies (panel K). The proteoliposomes prepared from RLIP76 purified from Caki-2 cells. Mean values ± SD for three experiments in triplicates are shown.