Identification of RKIP Ligands Figure 8. Compound 48 reduces cell viability in HeLa cells. HeLa cells (46104 cells/ml) expressing wild-type (open bars) or depleted RKIP (solid bars) were serum starved overnight and then pre-treated for 30 min with 20 mM DMSO (control) or compound 48. Reaction was stopped by the addition of cell titer blue (20 ml) to each well and incubated for 2 h at 37uC. Metabolic activity was measured on a fluorometer (Fluorescence 560Ex/590Em) and the results are described as the mean arbitrary units of fluorescence of triplicate wells of two independent experiments. **p,0.01 indicates the significance of the change relative to the corresponding sample in the absence of compound 48. doi:10.1371/journal.pone.0010479.g008 locostatin or 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (DHPE) and incubated for 30 min at 4uC. Complexes were washed three times with binding buffer with corresponding concentrations of RKIP binding compounds (98, 26 or 48) or locostatin or DHPE and boiled in sample buffer (2X). Lysates were separated on SDS-PAGE (12%), transferred to nitrocellulose and immunoblotted with anti-RKIP antibody. RKIP phosphorylation In vitro phosphorylation of RKIP by PKCa was as previously described [21]. Briefly, RKIP (5 mg) was combined with increasing concentrations of RKIP binding compounds (98, 26 or 48), locostatin or DHPE and the reaction was initiated with 100 mM ATP containing 5 mCi of [c-32P]ATP (Perkin Elmer) at 30uC for 10 min. The reaction was terminated using sample buffer (6X) and boiling at 100uC for 5 min. Lysates were separated on SDS-PAGE (12%), transferred to nitrocellulose and visualized by exposing to film. Results were quantified using phospho-imager screens (Molecular Dynamics) and analyzed with ImageQuant software [3]. MAPK activation assay HeLa cells (wild-type or RKIP depleted) were seeded in 6-well plates at 0.56106/ml and allowed to adhere overnight at 37uC [1]. Cells were serum starved overnight in DMEM media with no supplements. The next day, cells were treated with 0.1% DMSO (vehicle control), 20 mM locostatin or RKIP binding compounds (at indicated concentrations) for 25 min followed by EGF (10 ng/ml) for 5 min at 37uC. The reaction was terminated by placing plates on ice and washing with cold PBS (containing protease inhibitors). Cells were homogenized by cell scraping in RIPA lysis buffer supplemented with sodium orthovanadate (0.1 mM), sodium fluoride (0.5 mM) and protease inhibitor mixture tablet. Cell lysates were sheared five times in a 1 ml tuberculin syringe attached with a 21 G 619 mm needle, left on ice for 1 h and then spun at 16, 0006g for 15 min at 4uC. Cell lysates (30 mg) were resolved by SDS- PAGE (12.5%), transferred to nitrocellulose and analysed by Western blotting with anti-phospho ERK or anti-total ERK (Cell Signaling Technology, Inc) primary antibodies. Membranes were probed with anti-rabbit or –mouse IR dye secondary antibodies (LI- COR Biosciences). The amount of MAPK activity determined for each sample was normalized to total ERK in each sample. Digital analysis of immunoreactivity was done using LI-COR Biosciences Infrared Imaging System which is quantitative and independent of exposure time. Odyssey software (version 2.1; Lincoln, NE) was used for analysis of the immunoblots. Cell Viability assay Cell viability was determined as a measure of metabolic activity according to manufacturer’s protocol with minor modifications. Briefly, HeLa cells (wild-type or RKIP depleted) were seeded in 96-well plates at 46104/ml and allowed to adhere overnight at 37uC. The next day, cells were treated with 0.1% DMSO (vehicle control) or 20 mM each of locostatin or RKIP binding compounds for 30 min followed by 20 ml cell titer blue reagent resazurin (Promega) for 2 h at 37uC. Metabolic activity was measured using a fluorometer at 560Ex/590Em. Statistical analysis Differences between compound treated or untreated cells were compared using a two-tailed unpaired Student’s t-test. Supporting Information Figure S1 Overlay of 1H, 15N-HSQC spectra of RKIP in the absence (black) and presence (red) of a potential ligand that our PLoS ONE | www.plosone.org 12 May 2010 | Volume 5 | Issue 5 | e10479