Effect of mutations on GS-E affinity, ATPase activity. Effect of mutations on GS-E affinity, ATPase activity. Equal amounts of the purified protein (15 μg) was added to DNPSG-Sepharose-4B affinity-resin for 1 hour, and unbound RLIP76 was measured in the supernatant by ELISA assay (standard curve r2 0.99) and 1/unbound was used as for DNPSG affinity. DNPSG-stimulated ATPase activity of each mutant was measured as previously described (19). The ATP-dependent DNP-SG transport activity was determined after reconstituting the purified recombinant proteins into artificial asolectin-cholesterol liposomes as described previously (19). All results were normalized to control, wild-type RLIP76. Percent residual activity is shown (A). A plot of DNPSG affinity versus either ATPase or transport activity normalized to wild-type is presented (B). The effect of these mutations on GS-E binding was studied using the GSH-MCB fluorescent-conjugate. RLIP76 MEF−/− cells transfected with RLIP76-GFP vector were treated with 50 μmol/L MCB (which gives rise to a fluorescent GSH-conjugate in cells) for 20 minutes at 37°C prior to 4% paraformaldehyde fixation, followed by molecular interaction analysis using FRET using time-resolved confocal microscope MT 200 (from Picoquant GmbH) with pulsed diode laser excitations at 405 nm (for MCB donor) and 475 nm (for GFP donor). For MCB observation, 465 nm (10 nm bandwidth) interference filter crossed with 430-nm long path cutoff and for GFP observation 490- to 530-nm interference filter crossed with 500-nm long path filter was used. The histogram graph presents observed fluorescence lifetime measured for the respective images, MCB alone (green) and MCB + RLIP76-GFP (red; C, left). There is no significant change in the fluorescence life time cells transfected with the mutant of RLIP76 lacking GSH-binding site (ΔS234A /K237A; blue; C, right). The mutant RLIP76 cDNAs were cloned into pcDNA3.1 eukaryotic expression vector and successful transient transfection of RLIP76−/− MEFs was shown by IHC against RLIP76 with FITC-labeled secondary antibody (D, top row). Representative photomicrographs of RLIP76-mutant transfected cells showing the consequent partial restoration of EGF-rhodamine endocytosis are shown (D, bottom row). Sharad S. Singhal et al. Mol Cancer Ther 2011;10:16-28 ©2011 by American Association for Cancer Research