MEFs will be reported elsewhere. MEFs were immortalized with a temperature sensitive SV40 large T-antigen. RKIP+/+ MEFs were depleted by transduction with shRNA vectors using a pLKO.1 lentiviral vector as previously described [10]. These cells were maintained in culture medium (as described for HeLa cells under 200 mg/ml geneticin and/or 2.5 mg/ml puromycin selection at 33uC/5% CO2. NMR analysis 15N-Enriched RKIP was prepared by expressing the GST- RKIP fusion protein in M9 minimal media supplemented with 15N-ammonium chloride. After purification, RKIP was excised from the fusion protein using thrombin and concentrated. The NMR sample contained 100 mM 15N-RKIP in 50 mM Tris HCl buffer (pH 7.4) containing 100 mM NaCl prepared in 93% H2O and 7% D2O. 1H,15N-HSQC spectra were acquired at 30uC on a Varian Inova 600 spectrometer equipped with a cryogenic probe. NMR data were processed using the NMRPipe suite [23] and analyzed using the NMRView program [24]. Sequence-specific resonance assignments for RKIP have been published (BMRB accession code 6783) [16]. The weighted shift, ((1H shift)2+0.17(15N shift)2)1/2, was calculated following the method of Farmer et al. [25]. In vitro Raf and PKC 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 [12] 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 locostatin or 1,2- dihexanoyl-sn-glycero-3-phosphoethanolamine (DHPE).and incu- bated for 30 min at 4uC. Complexes were washed three times with binding buffer with corresponding concentrations of locostatin or DHPE and boiled in sample buffer (26). 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 [12]. Briefly, RKIP (5 mg) was combined with increasing concentrations of locostatin or DHPE and the reaction was initiated with 100 mM ATP containing 5 mCi of [c-32P]ATP at 30uC for 10 min. The reaction was terminated using sample buffer (66) 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. Membranes were subsequently immunoblotted with anti-RKIP antibody [4]. MAPK activation assay HeLa cells or MEFs (RKIP+/+ or RKIP2/2) were seeded in 6- well plates at 0.56106/ml and allowed to adhere overnight at 37uC or 33uC respectively [5,10]. Cells were serum starved Locostatin Impairs Migration overnight in DMEM media with no supplements. The next day cells were treated with DMSO (0.1 or 0.25%) or locostatin (20 or 50 mM) for 25 min followed by EGF (10 ng/ml) for 5 min at 37uC or 33uC respectively. 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 G619 mm needle, left on ice for 1 h and then spun at 16, 0006g for 10 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 (Cell Signaling Technology, Inc) or anti-total ERK (Cell Signaling Technology, Inc) primary antibodies. Membranes were probed with anti-rabbit and –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. Scratch wound assay MEFs of varying genotypes (as indicated) were seeded at 0.56106/ml in 6-well plates under selection as described above and allowed to adhere for 24 h at 33uC. On the day of the assay wells were rinsed once with warm PBS and then kept in supplemented DMEM media in the absence or presence of DMSO (0.1 or 0.25%) or locostatin (20 or 50 mM) at 33uC for 24 h. Wounding was performed using a pipette tip and by drawing four wounds of standard size across a confluent monolayer of cells. Prior to incubation at 33uC/5% CO2, digital images were taken at time zero followed by four, eight and 24 h. Images were taken on a phase-contrast microscope (Leica DMIRB, epifluorescence) at 65 magnification. Wound area was determined as a percentage of the wound size at time zero and using the area of the wound as measured by Image J software (NIH). Initial wound area was standardized for all conditions tested and then changes in area over time were expressed as a percentage of the initial time zero wound. Actin localization and mitotic spindle microscopy MEFs of varying genotypes (as indicated) were plated at 26104 cells per 15 mm coverslip and grown for 48 h. Cells were treated with DMSO only (Control), or locostatin (20 or 50 mM), or paclitaxel (10 mM) for 1 to 6 h (as indicated), followed by fixation with 4% paraformaldehyde (in 0.1 M Na-phosphate, pH 7.4) for 10 mins at room temperature (RT), permeabilized with 0.1% TritonX-100 in PBS for 2 mins at RT, and blocked with 3% BSA in PBS overnight at 4uC. Alpha-tubulin was detected using a mouse antibody at 1:50 dilution and actin was detected using Alexafluor 594-tagged phalloidin (4 U/ml) in 1% BSA in PBS for 2 h at RT. The cells were washed 3 times with 1% BSA in PBS, and the tubulin antibody detected with FITC-conjugated donkey anti-mouse antibody at 1:100 in 1% BSA in PBS for 1 h at RT. The cells were then washed 3 times with PBS. The second PBS wash contained 1 mg/ml Ho¨echst 33342 to visualize DNA/ chromatin. The coverslips were mounted on slides using Vectashield Hardset. Digital photomicrographs were captured using Openlab Darkroom (Improvision) and a Retiga 1300 color digital camera (Q Imaging). PLoS ONE | www.plosone.org 12 June 2009 | Volume 4 | Issue 6 | e6028