the manufacturer’s instructions. The column was sequentially washed with 10 ml buffer A, 10 ml buffer A supplemented with 0.5% NP-40, 10 ml buffer A supplemented with 500 mM NaCl and 10 ml buffer A. Glu-Glu-SOS was eluted by applying buffer A containing 50 mg/ml EYMPME peptide. SOS fractions were pooled, concentrated, dialyzed against 50 mM Tris pH 7.5, 50 mM NaCl, 1 mM DTT and 50% glycerol, and stored at 220uC. Kept in this way, SOS retained nucleotide exchange activity towards recombinant H-Ras for at least two weeks. BrdU-based proliferation assay NIH 3T3 cells expressing dox-inducible ezrin wild type or R40L mutant were plated at low cell density on coverslips placed in 12- well plates, followed by incubation with 20 mM/well bromodeox- yuridin (BrdU, Oncogene) for 3 h. Cells were fixed with 4% paraformaldehyde in ‘cytoskeleton buffer’ (CB: 10 mM PIPES, 150 mM NaCl, 5 mM EGTA, 5 mM glucose and 5 mM MgCl2, pH 7.0) at room temperature (RT) for 20 min, washed three times in PBS and permeated in 0.1% Triton-X-100 in PBS for 1 min. After blocking in 1% BSA for 1 h, the coverslips were treated with a cocktail of 2 mg/ml DAPI (Invitrogen), 250 mg/ml a-BrdU/ Fluorescein (Alexis), 106 DNase-Buffer, DNase and 200 mg/ml a- myc/TRITC for 1 h. Cells were washed three times in PBS, mounted on glass slides with 10 ml Prolong Antifade reagent (Molecular Probes) and images obtained using epifluorescence with an Axiovert 135 TV microscope (Zeiss). Soft agar assay RT4 stably transfected cells were detached from culture plates with 0.25% trypsin, resuspended in complete medium and counted. Cells (16104) were resuspended in 3.6 ml of medium containing 10% FCS. This mastermix was split into two, doxycycline (1 mg/mL) was added to one half and to each mixture 200 ml of warm agar solution (stock is 3.3% in sterile PBS) was added and mixed. The mixture was rapidly dispensed into 24-well plates at 500 ml volume and cooled for 2 min at 4uC. Colonies were counted after 10 to 14 days. siRNA experiments Single siRNA oligonucleotides were purchased from Ambion, used siRNA sequences can be found in the Supplementary methods. In brief 46105 cells were seeded overnight in a 10 cm plate. Transfection of all siRNAs was performed using lipofecta- mine according to manufacturer’s instructions. The following day cells were given fresh medium and left to recover for at least 8 h. Cells were serum starved overnight prior to 10 ng/ml PDGF stimulation for 5 minutes. siRNA with exogenous reconstitution of human ezrin, radixin and moesin NIH 3T3 cells were transfected with ERM siRNAs using lipofectamine. The following day cells were given fresh medium and left to recover for at least 8 h. Cells were then transfected with plasmid DNA containing human ezrin, radixin and moesin using lipofectamine according to manufacturer’s instructions. Cells were serum starved overnight prior to 10 ng/ml PDGF stimulation for 5 min. Affinity precipitation and immunoprecipitation For pull-downs with GST-Raf1-RBD, cells were lysed in 25 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1% Igepal CA-630, 10% glycerol, 1 mM EDTA, 1 mM sodium vanadate, 1 mM PMSF, 10 mg/ml aprotinin and 10 mg/ml Ras Requires ERMs leupeptin. For affinity precipitation supernatants were incubated with 5 mg recombinant fusion protein rotating at 4uC for 1 h. Ras assays were performed on pooled clones expressing ezrin wild type compared to ezrin R579A mutant. 46105 cells were seeded overnight in a 10 cm plate. Cells were serum starved overnight prior to 10 ng/ml PDGF stimulation for 3 min. For pull-downs with GST-Nterminal ezrin or GST-Grb2, cells were lysed in 25 mM HEPES pH 7.5, 20 mM NaCl, 10 mM MgCl2, 0.5% Lubrol 17A17, 1 mM sodium vanadate, 1 mM PMSF, 10 mg/ml aprotinin and 10 mg/ml leupeptin. Supernatants were incubated with either 5 mg recombinant fusion protein rotating at 4uC overnight. For all pull-downs 46105 cells were seeded overnight in a 10 cm plate. For immunoprecipitation procedure as in Morrison et al., 2001 [16]. In brief, 26106 cells were seeded overnight in a 20 cm plate. Cells were serum starved overnight prior to 10 ng/ml PDGF stimulation for 1 minute. Cells were lysed in 25 mM HEPES pH 7.5, 20 mM NaCl, 0.5% Lubrol 17A17, 1 mM sodium vanadate, 1 mM PMSF, 10 mg/ml aprotinin and 10 mg/ml leupeptin. Supernatants were incubated with 2 mg of antibody rotating at 4uC for 1 h. SDS-PAGE, immunoblotting and coomassie staining Following electrophoresis with 7.5–12.5% SDS-PAGE gels, the proteins were transferred to PVDF membranes (Millipore) and treated with blocking buffer (5% skimmed milk, 0.1% Tween-20, 10 mM Tris at pH 7.6 and 100 mM NaCl) at RT for 1 h. The membranes were then incubated with primary antibody in blocking buffer for another hour at RT or overnight at 4uC. After three washes, the membranes were incubated with secondary antibody at RT for 1 h, then developed and visualized using enhanced chemiluminescence (Amersham). For coomassie-based gel staining, Imperial protein stain solution (ThermoScientific) was used. Immunoblotting quantification was performed using Quan- tity One software (Bio-Rad Laboratories). Note: immunoblots for aERM (unless otherwise stated) a mixture of ezrin (C-19), radixin (C-15) and moesin (C-15) was used. For a single ezrin blot, the 3C12 antibody was used. Pull-down assays using recombinant proteins Ras/ezrin. Ras/ezrin interaction was tested for different conditions (GTPcS-Ras vs. GDP-Ras) and different purified ezrin protein mutants. Ras loading with nucleotides was performed as described in Margarit et al., 2003 [43]. The respective His-tagged ezrin protein (1–2 mg) was linked to Ni-Sepharose and incubated with 50–100 ng loaded Ras in 500 ml interaction buffer (50 mM HEPES pH 7.5, 50 mM NaCl, 10 mM MgCl2 and 0.5% Triton- X-100) overnight at 4uC under constant rotation. The following day, the probes were washed four times with 100 ml ice-cold washing buffer (50 mM HEPES pH 7.5, 80 mM NaCl, 10 mM MgCl2 and 0.5% Triton-X-100), with a short centrifugation step between the washings. During the final washing step, the washing buffer was completely removed and the remaining proteins were subjected to SDS-PAGE and immunoblotting. SOS/ezrin: SOS/ ezrin interaction studies were performed either in the presence or absence of F-actin/G-actin. G-actin (Cytoskeleton) was polymerized to F-actin following the manufacturer’s protocol. Interactions between SOS and ezrin, and F-actin/G-actin were then carried out either in F-actin-Buffer (5 mM Tris pH 8.0, 0.2 mM CaCl2, 50 mM KCl, 2 mM MgCl2, 1.2 mM ATP and 0.5 mM DTT) or G-actin-Buffer (5 mM Tris pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP and 0.5 mM DTT) following the procedure above. PLoS ONE | www.plosone.org 12 November 2011 | Volume 6 | Issue 11 | e27511