obtained by subcloning into pSCherry2 (Eurogentec, Seraing, Belgium). The flot-1 (NM_022701.2) constructs in pGBKT7 vector have been described previously [24,52]. Rat flotillin-1 in pET41a was generated by PCR cloning. CAP-Flag (murine isoform 1, GenBank accession number: U58883) and SoHo-BD were a kind gift of Dr. A. Saltiel. Other CAP constructs were generated using PCR. FGFR-Myc construct was a kind gift of I. Kovacevic (University of Frankfurt). Antibodies Rabbit polyclonal antibodies against FRS2 (Western blotting) and ERK1/2 and the monoclonal antibody against pERK1/2 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibodies against Flag tag and FRS2 (for immunoprecipitation and immunofluorescence) were from Sigma- Aldrich (Taufkirchen, Germany). Rabbit polyclonal antibodies against Akt, phospho-Akt (Ser473) and phospho-Tyr were from Cell Signaling Technology (Danvers, MA, USA). Monoclonal antibodies against flot-1 and flot-2 were from Transduction Laboratories (Franklin Lakes, NJ, USA) and anti-CAP was from Upstate (Lake Placid, NY, USA). A mouse monoclonal antibody against GAPDH was from Biozol (Eching, Germany). The primary antibodies used for immunofluorescence were detected with a Cy3-conjugated goat anti-mouse antibody (Jackson Immunoresearch, West Grove, PA, USA) and with an Alexa Fluor 488 donkey anti-rabbit antibody (Molecular Probes, Karlsruhe, Germany). Secondary antibodies goat anti-mouse and goat anti-rabbit coupled to horseradish peroxidase (HRP) were from Southern Biotechnologies (Birmingham, AL, USA) and Zymed (Invitrogen, Karlsruhe, Germany), respectively. Yeast-two-hybrid analysis 50 ml YPDA was inoculated with several fresh colonies of the yeast strain AH109, and the culture was incubated with shaking at 30uC for 16 h to stationary phase (OD600.1.5). An aliquot of the culture was transferred to 300 ml YPDA medium and incubated at 30uC with shaking until OD600 0.560.1. Preparation of competent cells and cotransformation with bait and prey constructs were done according to the manufacturer’s protocol (Matchmaker GAL4 Two-Hybrid System 3, Clontech). Transfor- mants were plated on SD/-Leu/-Trp plates for selection. After a few days, colonies were picked and transferred onto SD/-Ade/ -His/-Leu/-Trp/X-a-gal plates for the detection of galactosidase activity and onto SD/-Leu/-Trp plates for growth control. Plates were photographed 3 days after plating. Preparation of yeast lysates Yeast cell pellet from a 2.5 ml overnight culture was washed and snap-frozen in liquid nitrogen. The pellets were resuspended in 100 ml cracking buffer (8 M urea, 5% w/v SDS, 40 mM Tris- HCl, 0.1 mM EDTA, 0.4 mg/ml bromphenolblue) supplemented with protease inhibitor cocktail, 5 mM PMSF and 10% b- mercaptoethanol. Thereafter, 100 ml of glass beads were added into the mixture which was vigorously vortexed and incubated for 10 min at 70uC. Insoluble material was pelleted by centrifugation at 21 900 g for 5 min. The supernatant was kept, while the insoluble pellet was further cooked for 5 min and then centrifugated for 5 min at 21 900 g. The supernatants of these two extractions were pooled and analyzed by Western blot. Cell culture and transfection HeLa cells were cultured at 8% CO2 and 37uC in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Karlsruhe, FRS2 Interaction with Flotillin-1 and CAP Germany), containing high glucose, 10% fetal calf serum (FCS, Invitrogen), 100 U/ml penicillin and 100 mg/ml strepto- mycin (Sigma-Aldrich, Taufkirchen, Germany). Hep3B cells were maintained in the same medium but at 5% CO2. For transient transfections, Hela cells were seeded out on 6 well plates and grown to a confluency of 90%, after which they were transfected with 1 mg plasmid DNA using Lipofectamine 2000 (Invitrogen). GST-protein expression The following bacterial strains were used for the expression of fusion proteins: BL21 transformed with either pGEX-4T-1 or FRS2-GST constructs and the strain Rosetta transformed with either pET41a, full length CAP-GST, flot-1-GST or one of the deletion constructs of CAP. The bacteria were grown at 37uC until OD600 0.4–0.6 and then induced with 1 mM IPTG for 5 h at 37uC, or with 50 mM IPTG over night at 16uC (in case of flot-1- FL-GST and flot-1-CT-GST). The cells were pelleted and lysed in lysis buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 1 mM EDTA, 5% glycerol, 0.1% NP-40) supplemented with with 100 mg/ml lysozyme, 1 mM PMSF, 1 mM dithiothreitol (DTT) and 1 mM aprotinin, leupeptin and pepstatin. GST proteins from the lysates were allowed to bind to glutathione-sepharose beads (GE Healthcare), washed with PBS and left on the beads for pull- downs. His-tagged protein expression and purification The bacterial expression strain SE1 transformed with FRS2- His was grown and induced as above. The bacteria were lysed in His-lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 5% glycerol, 0.1% NP-40) containing 100 mg/ml lysozyme, 1 mM PMSF, 5 mM imidazole, 2 mM b-mercaptoethanol and 1 mM of each of the protease inhibitors (aprotinin, leupeptin, pepstatin). FRS2-His was bound to Ni-NTA agarose beads (Qiagen, Hilden, Germany). The beads were washed with the lysis buffer supplemented with increasing concentrations of imidazole, and FRS2-His was eluted from the beads with lysis buffer containing 250 mM imidazole. To remove excess salt and imidazole, which might interfere with binding in further experiments, the protein was dialysed against the dialysis buffer (50 mM Tris-HCl pH 7.6, 300 mM NaCl, 1 mM EDTA, 1 mM DTT, 20% glycerol) at 4uC over night. GST-pulldowns HeLa or Hep3B cells were lysed for 30 min on ice in lysis buffer (50 mM Tris pH 7.4, 0.15 M NaCl, 2 mM EDTA, 1% NP-40) supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich). Cell lysates were incubated with either GST or GST-tagged proteins immobilized on glutathione-sepharose beads over night at 4uC. The beads were washed three times with 1 ml lysis buffer. The samples were resuspended in loading buffer containing 50 mM DTT, boiled 5 min at 94uC and separated by SDS- PAGE. GST pulldown using purified proteins Direct GST pulldown experiments were performed on ice for 3 h with flicking, using 5 mg of the purified proteins (GST, CAP- GST, FRS2-His). The beads were washed three times with a buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.01% Triton X-100, resuspended in 26 SDS sample buffer containing 50 mM DTT, heated for 5 min at 94uC and separated by SDS-PAGE. PLoS ONE | www.plosone.org 10 January 2012 | Volume 7 | Issue 1 | e29739