Identification of RKIP Ligands analysed by immunoblotting with anti-phospho ERK and anti-total ERK antibodies. ERK phosphorylation was assessed by normalizing phospho-ERK levels to total ERK levels as depicted in line graphs. The results shown are mean 6 range of two independent experiments. doi:10.1371/journal.pone.0010479.g006 Materials and Methods Reagents and antibodies A compound library containing 10, 000 drug-like chemicals was purchased from ChemBridge. N’-(2,4-dinitrophenyl)-2,3,4,5,6- pentafluorobenzohydrazide (compound 26), N- {[(2-bromo-4- nitrophenyl)amino]carbonothioyl}-3-(2-thienyl)acrylamide (com- pound 48), 4-{4-[(2,4-dinitrophenyl)amino]phenyl}amino)-4-oxo- 2-butenoic acid (compound 98) were from ChemBridge Corpo- ration. (S)-(+)-4-benzyl-3-crotonyl-2-oxazolidinone (locostatin) used for NMR analysis was from Aldrich. Protease inhibitor cocktails and locostatin unless otherwise indicated were from Calbiochem. All cell culture media and supplements were from GIBCO (Invitrogen). 15N-ammonium chloride was from Cam- bridge Isotope Laboratories. Anti-phospho and -total ERK antibodies were from Cell Signaling Technology. Anti-rabbit and –mouse IR dye secondary antibodies as well as Odyssey blocking buffer used for Western blotting were purchased from LI- COR Biosciences. Anti-RKIP antibody was developed as described [3]. IgG sepharose beads were from Amersham Biosciences (GE Healthcare). Biotinylated thrombin (50 U) was from Novagen. Cell lines and cell culture HeLa cells stably expressing short hairpin RNA vectors for depleting human RKIP or rat RKIP control (wild-type RKIP) were maintained at 37uC/5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 50 U/ml penicillin and 50 mg/ml streptomycin under puromycin (2 mg/ml) selection as previously described [1]. Protein expression BL21(DE3) cells (Novagen) [31] harboring a pMCSG7-derived expression vector [32] for rat RKIP were grown at 37uC in the M9 media with15N-NH3Cl as the sole nitrogen source and supple- mented with ampicillin (100 mg/ml). The expression construct consisted of a His6 tag fused in frame with a TEV protease cleavage signal and the entire open reading frame (residues 1-187) of wild-type rat RKIP (rRKIP). Protein expression was induced with 0.2 mM isopropyl-1-thio-b-d-galactoside (IPTG) when the optical density at 600 nm of cultures reached ,0.7, and expression was allowed to proceed for 16 hours at 30uC. The His-tagged rRKIP expressing cells were harvested by centrifuga- tion and stored at 280uC. Protein purification Cells having expressed His-tagged rRKIP were lysed in 50 mM Tris-HCl pH 8.0 by a combination of hen egg lysozyme (0.5 mg/ ml) (Sigma) and sonication. The lysate was cleared by centrifu- gation. Concentrated NaCl was added to the lysate at a final concentration of 0.5 M. The solution was loaded onto a Ni-affinity column (Ni Sepharose 6 Fast Flow; Amersham) equilibrated with Column Wash Buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl). The column was washed with 10 column volumes of Column Wash Buffer followed by 5 column volumes of Column Wash Buffer containing 20 mM imidazole. His-rRKIP was eluted from the column with Column Wash Buffer containing 500 mM imidazole. Fractions containing His-rRKIP were pooled, dialyzed into 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 1 mM DTT and cleaved overnight with his-tagged TEV protease to remove the N- terminal His6 tag by the protocol previously described [33]. The cleaved protein was purified from the protease and the cleaved affinity tag by passing the cleavage product over the nickel-affinity column and collecting the column flow-through containing the purified protein. Purity was found to be .95% by SDS-Page analysis with yields routinely exceeding 50 mg/L of culture. Samples for NMR analysis of ligand binding contained 75 mM 15N-rRKIP in 50 mM Tris-HCl pH 7.4 containing 100 mM NaCl and 7% D2O. Ligand stocks were prepared in the same buffer and the pH value adjusted appropriately. NMR Spectroscopy 1H,15N-HSQC spectra [34] of 15N-rRKIP in the presence of the various ligands were acquired for 40 min at 30uC on a Varian Inova 600 spectrometer equipped with a cryogenic probe and a 9- position sample changer. NMR spectra were processed using the NMRPipe suite [35]. Spectra were analyzed and peak list for apo and ligand bound samples were generated using NMRView [36]. Exact peak positions and intensities were obtained using 2- dimensional deconvolution of peak positions with the pkfit program. Global fitting analysis of peak position and intensity was performed using the program GLOVE (pkfit and GLOVE were developed by the Peter E. Wright laboratory, The Scripps Institute) modified to fit the data to a single-site binding model. Errors were estimated through 500 Monte Carlo simulations of the globally fitted data. The residue specific amide proton pKa values were determined by fitting pH dependent changes of the amide 1H chemical shifts to the Henderson-Hasselbach equation using SigmaPlot 8.0 (Jandel Scientific). Compound screening was performed in a two-step manner as follows. In the first step, 2 ml each of 30 compounds (dissolved in DMSO at the concentration of 5 mg/ml) were pooled and added to 540 ml of 50 mM Tris-HCl pH 7.4 containing 100 mM NaCl, 7% D2O and 50 mM 15N-RKIP. The final concentration of each compound (17 mg/ml) corresponds to 40–50 mM. The 1H,15N- HSQC spectra for RKIP with each pooled compound were collected and compared with that of RKIP with DMSO (negative control). The concentration (10%) of DMSO used in the initial screen did not significantly alter the HSQC spectrum, indicating that RKIP was stable in the presence of 10% DMSO. Hit compounds were identified by individually analyzing each of the compounds in those initial mixtures that gave significant HSQC perturbation. In vitro Raf kinase assay The pRav-Flag-Raf-1 plasmid (TAP-Raf-1; Tandem Affinity Purification using Protein A and Flag tagged-Raf-1) was constructed as previously described [21] and stably expressed in H19-7 cells. Briefly, TAP-Raf-1 was immunoprecipitated from H19-7 cells that were serum starved overnight in DMEM media with no supplements, and TAP-Raf was isolated on IgG sepharose beads. Cells were lysed in TAP-lysis buffer (10 mM HEPES pH 7.4, 3 mM MgCl2, 10 mM KCl, 5% Glycerol, 0.1% NP40) and cleared by centrifugation. Cell lysates were combined with pre-equilibrated TAP-lysis buffer IgG sepharose beads and incubated for 1 h at 4uC. Beads were washed with TAP-lysis buffer and equilibrated with binding buffer (10 mM HEPES pH 7.4, 3 mM MgCl2, 10 mM KCl, 150 mM NaCl). Beads were aliquoted and combined with RKIP (5 mg) and increasing concentrations of RKIP binding compounds (98, 26 or 48), PLoS ONE | www.plosone.org 10 May 2010 | Volume 5 | Issue 5 | e10479