Fluorescence microscopy Cells were grown on coverslips placed in six-well plates. Cells were fixed with 4% paraformaldehyde in ‘‘cytoskeleton buffer’’ (CB: 10 mM PIPES, 150 mM NaCl, 5 mM EGTA, 5 mM glucose, 5 mM MgCl2, pH 7.0) at room temperature for 20 min, washed 3 times in CB and permeated in 0.05% Triton- X-100 in PBS for 1 min. Actin fibers were detected by phalloidin conjugated to Alexa 594 (Molecular Probes) for 1 hour at a dilution of 1:50. Cells were washed 3 times, mounted on glass slides with 10 ml Prolong Antifade reagent (Molecular Probes) and images obtained using epifluorescence with an Axiovert 135 TV microscope (Zeiss) equipped with a Plan-Apochromat 1006/1.40 oil immersion objective. Images were recorded with a cooled, back-illuminated CCD camera (TE/CCD-1000 TKB, Princeton Instruments) driven by IPLab Spectrum software (Scanalytics). GEF activity measurement – streptolysin O Intracellular GEF activity was measured as described in de Vries-Smits et al., 1995 and Stephens et al., 1993. In brief, NIH 3T3 cells were seeded in 6-well plates at 66103 cells/cm2. Transfection of siRNA duplexes or DNA constructs expressing ezrinWT, R579A was performed the next day (the R40L a dox inducible mutant cell line was used). Another day later, cells were deprived of serum overnight to perform the assay after 48 h of ERM knock-down or construct expression (dox added were indicated). Assay kinetics was started after culture medium was replaced with 600 ml permeation buffer (50 mM HEPES pH 7.5, 107 mM K-glutamate, 23 mM NaCl, 3 mM MgCl2, 0.3 mM CaCl2, 1 mM EGTA, 1 mM ATP, 30 U/ml streptolysin O (Aalto Bioreagents), 100 mCi/ml [a32P]-GTP (Hartmann Analytik) and 2/+ 10 ng/ml PDGF). At indicated time points, the exchange reaction was stopped by adding 1 ml lysis buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1% NP-40, 100 mM GDP, 100 mM GTPcS, protease inhibitor cocktail ‘complete w/o EDTA’ (Roche) and 5 mg/ml anti-Ras antibody Y13-259). Lysates were cleared by centrifugation, supplemented to contain 500 mM NaCl, 0.6% sodium deoxycholate and 0.06% SDS, and Ras was immunoprecipitated with protein Gplus agarose (Oncogene). The precipitates were washed six times with 1 ml Ras wash buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM MgCl2, 0.1% Triton-X-100 and 0.005% SDS) and subjected to Cherenkov counting. GEF activity measurement – SOS containing complexes from cells Measurement of nucleotide exchange activity of SOS towards Ras was performed as described in Downward et al., 1995 and Rubio et al., 1999. In brief, four 15 cm plates were used. In each plate 26106 dox inducible NIH 3T3 cells were seeded (dox inducible for ezrinWT or ezrinR579A mutant). The next day cells were serum starved and dox added overnight. Following day, in each plate cells were lysed in 1 ml lysis buffer after 10 ng/ml PDGF stimulation (50 mM HEPES pH 7.4, 0.5% Lubrol 17A17, 100 mM NaCl, 5 mM sodium vanadate, and protease inhibitor cocktail ‘complete w/o EDTA’). The lysate was mixed and then divided again into four and SOS was pulled down for 2 h using GST-Grb2. Equal amount of SOS and the presence of ezrin were confirmed by immunoblotting. The pulldowns were stored on ice until used in the exchange assay. Ras loading: Recombinant H- Ras (50 ng) was loaded at 37uC for 5 min with 25 mCi [a32P]- GTP in 100 ml loading buffer (50 mM HEPES pH 7.5, 5 mM EDTA and 5 mg/ml BSA). The reaction was stopped by adding 5 ml 0.5 M MgCl2 on ice. High-magnesium buffer (800 ml) Ras Requires ERMs (50 mM HEPES pH 7.5 and 5 mM MgCl2) and 1 mM cold, non-radioactive GTP were added, and MgCl2 was adjusted to 5 mM. This mixture (25 ml) was added to the immunoprecipitates and nucleotide exchange was assayed at 30uC for 30 min. The reaction was stopped by placing on ice and adding 300 ml Ras wash buffer, 1 mg/ml BSA and 5 mg Y13-259 anti-Ras antibody. Ras was immuno-precipitated with protein Gplus agarose. The precipitates were washed four times with 1 ml Ras wash buffer and subjected to Cherenkov counting. Protein purification from Escherichia coli The expression vector pET15TEV fuses an N-terminal polyhistidine tag to the protein followed by a tobacco etch virus (TEV) cleavage site. E. coli cells (Rosetta 2 DE3, Novagen) were transformed with SOS, ezrin or Ras protein expression constructs and grown in LB medium supplemented with antibiotics (100 mg/ l ampicillin and 100 mg/l chloramphenicol; Carl Roth). Protein expression was induced by 100 mM IPTG (for Ras; Carl Roth), 250 mM IPTG (SOS) or 500 mM IPTG (ezrin) at a cell density corresponding to an absorbance of 0.4–0.6 at 600 nm, and the proteins were expressed at 33uC overnight (Ras), at 30uC for 6 h (SOS) or at 37uC for 4 h (ezrin). Cells were collected by centrifugation for 20 min at 6,0006g, resuspended in either Ras resuspension buffer (RB; 25 mM Tris pH 7.4, 2.5 mM MgCl2, 100 mM GDP and 0.5% Triton-X-100), SOS resuspension buffer (50 mM Tris pH 7.4, 1 M NaCl and 0.5% Triton-X-100) or ezrin resuspension buffer (50 mM Tris pH 8.0 and 150 mM NaCl) containing protease inhibitors and snap frozen in liquid nitrogen. After thawing, lysozyme (Carl Roth) was added. Cell lysis was carried out by ultrasonification (15 sec impulse, 30-sec break). Cell debris was collected by centrifugation at 15,0006g in a JA 25.50 rotor for 10 min. All purifications were carried out at 4uC. The remaining clear lysate was incubated with Ni-Sepharose 6 Fast Flow (GE Healthcare) overnight and transferred to Glass Econo- Column Columns (Bio-Rad). After several washing steps with RB and RB plus 30 mM imidazole, proteins were eluted using 250 mM imidazole in the corresponding RB (including 140 mM NaCl for Ras). Fractions containing proteins were pooled and dialyzed against buffer: Ras (50 mM Tris pH 7.4, 5 mM MgCl2 and 1 mM DTT); SOS (20 mM Tris pH 8.0 and 100 mM NaCl); and ezrin (50 mM Tris pH 8.0 and 150 mM NaCl). SOS and Ras proteins were aliquoted, snapped frozen and stored at 280uC. Ezrin proteins were either kept at 4uC or 220uC. When required, the His-tag was cleaved using TEV Protease (Promega) following the manufacturer’s instructions. Protein purification from insect cells Full-length SOS1 was prepared essentially as described in [42]. Briefly, Sf9 cells (Merck, Biosciences, Germany) infected in triple- bottom culture flasks with a baculovirus encoding the sequence of full-length human SOS1 in fusion with an N-terminal Glu-Glu epitope tag (EYMPME) (cloned in transfer vector pAcC13, kind gift of Frank McCormick, San Francisco) were harvested 72 h post infection. Cells were washed once in PBS, resuspended in 10 ml ice-cold hypotonic solution (50 mM Tris pH 7.5, 5 mM MgCl2, 1 mM DTT and protease inhibitors)/ml cell pellet and placed on ice for 10 min. Cells were lysed with 20 strokes in a Dounce homogenizer on ice and insoluble debris was removed by centrifugation at 100,0006g for 30 min at 4uC. The supernatant was loaded onto a 1–2-ml anti-Glu-Glu column pre-equilibrated in buffer A (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT and protease inhibitors), previously prepared by coupling 1 mg anti-Glu-Glu antibody (kind gift of Julian Downward, London) to 1 ml Actigel matrix (Sterogene) following PLoS ONE | www.plosone.org 11 November 2011 | Volume 6 | Issue 11 | e27511