Stable knockdown of flotillins using lentiviruses Hep3B cells were seeded on a 96-well plate at a density of 32 000 cells/well one day prior to transduction. The cells were infected with MISSION shRNA Lentiviral Transduction Particles (Sigma-Aldrich) with a multiplicity of infection of 5.6. Hexadi- methrine bromide (100 mg/ml) was used to increase the efficiency of infection. Knockdown of flot-2 was achieved by using viral clones TRCN0000149396 and TRCN0000150223, whereas for the knockdown of flot-1, clones TRCN0000029310 and TRCN- 0000029309 were used. Viruses were removed one day after infection. Selection for stable cell lines was started two days post- infection using 2 mg/ml of puromycin. Transient knockdown of flot-1 using siRNA oligos HeLa cells were grown on 12 well plates to a confluency of 80% and transfected with 85 nM siRNA oligonucleotide duplexes targeting flot-1 (StealthTM siRNA; Invitrogen) using Lipofectamine 2000 (Invitrogen). As a control, an oligo which does not target any human sequence (StealthTM RNAi Negative Control Duplexes medium GC, Invitrogen) was used. These duplexes have previously been well characterized by us [25,27] and routinely result in more than 80% knockdown of flot-1 in about 95% of the cells. No off-target effects have been detected so far. Growth factor treatment Hep3B cells were starved 18 h prior to treatment with 100 mM sodium pervanadate for 30 min. HeLa cells were serum starved for 24 h and then treated with 50 ng/ml bFGF (Sigma-Aldrich) and 10 mg/ml heparin. Immunofluorescence Hep3B cells were grown on coverslips and fixed with ice-cold methanol. Unspecific binding was blocked with 1% BSA/PBS, and the cells were labeled with primary antibodies for 1 h and stained with Cy3 and AlexaFluor 488 conjugated secondary antibodies for 45 min. The samples were embedded in Gelmount (Biomeda, Foster City, CA, USA) supplemented with 1,4- diazadicyclo(2,2,2)octane (DABCO, Fluka, Neu-Ulm, Germany). Images were taken with a confocal laser-scanning microscope (Zeiss LSM510 Meta). Coimmunoprecipitation Cells were lysed for 30 min on ice in coimmunoprecipitation buffer (100 mM Tris pH 8.0, 0.15 M NaCl, 1 mM MgCl2, 1% Triton X-100), supplemented with Protease Inhibitor Coctail, 60 mM n-octyl-b-D-glucopyranosid (AppliChem) and 1 mM vanadate. Mouse tissues were grinded in liquid nitrogen. 25 mg of powdered frozen tissue were homogenized in a tissue lyzer (Retsch, Germany) in 500 ml coimmunoprecipitation buffer. The lysates were precleared three times with 50 ml Pansorbin beads (Calbiochem, Nottingham, UK). Precipitation was performed using antibody-coupled Protein A Dynabeads (Invitrogen) for 16 h at 4uC. The beads were washed three times with lysis buffer. Precipitated proteins were solubilized in loading buffer containing 50 mM DTT and boiled 5 min at 94uC. Proteins were separated by SDS-PAGE and subjected to immunoblotting with specific antibodies. References FRS2 Interaction with Flotillin-1 and CAP Preparation of detergent insoluble membranes Hep3B cells were grown in 15 cm dishes, starved overnight and treated or not with 100 mM sodium pervanadate for 30 min. Lipid rafts were prepared using density gradient centrifugation as described [40]. 1.2 ml fractions of the gradients were collected, supplemented with SDS (final concentration 2%) and mixed with 46 loading buffer containing 100 mM DTT and 20% b- mercaptoethanol. The samples were boiled for 5 min at 94uC. Equal volumes were analyzed by SDS-PAGE and Western blotting. In order to detect the ganglioside GM1, 5 ml of the fractions were slot-blotted onto nitrocellulose and incubated with 0.5 mg/ml cholera toxin subunit B (CTX-B; Invitrogen, Karls- ruhe, Germany) conjugated with HRP. Statistics Unless otherwise stated, all experiments were performed at least three times. For the statistical analysis, Western blots were quantified by scanning densitometry using Quantity One Software (Biorad, Munich, Germany). Data are shown as mean 6 SD. Statistical comparisons between groups were made using one way ANOVA analysis (Bonferoni test) using GraphPad Prism 4 software. Values of p,0.05 were considered significant (*) while values of p,0.01 and p,0.001 were defined very significant (**) and extremely significant (***), respectively. Supporting Information Figure S1 Expression of the proteins during yeast two- hybrid analysis and analysis of knockdown efficiency of flotillins in the stable Hep3B cells. (A) Flot-1 and FRS2 domain constructs used for identification of the interacting domains in yeast two-hybrid assay. (B) Yeast lysates were prepared from the transformed cells used for the yeast two-hybrid analysis, and the expression of the fusion proteins was detected. Autoactivation test was done by plating yeast colonies transformed with the bait onto the selective plate containing the substrate of a- galactosidase (–T+a-X-Gal plate). (C) Western blot analysis of knockdown efficiency of flot-1 and flot-2. GAPDH was used as an equal loading control. (D) Immunofluorescence staining of endogenous flotillins in control (left column), flot-2 knockdown (upper right) and flot-1 knockdown (lower right) cells. Scale bars 10 mm. (TIF) Acknowledgments We wish to thank Ivan Dikic, head of the Institute of Biochemistry II, University Clinic of Frankfurt, for allowing us to use the confocal laser scanning microscope and yeast cell culture facilities. We would like to thank Koraljka Husnjak for help concerning yeast two-hybrid analysis and GST purification, and Ralf Fu¨llkrug for his superb technical assistance. Antje Banning is acknowledged for the critical reading of the manuscript. Author Contributions Conceived and designed the experiments: AT RT. Performed the experiments: AT ST RT. Analyzed the data: AT RT. Wrote the paper: AT RT. 1. Kouhara H, Hadari YR, Spivak-Kroizman T, Schilling J, Bar-Sagi D, et al. (1997) A lipid-anchored Grb2-binding protein that links FGF-receptor activation to the Ras/MAPK signaling pathway. Cell 89: 693–702. 2. Meakin SO, MacDonald JI, Gryz EA, Kubu CJ, Verdi JM (1999) The signaling adapter FRS-2 competes with Shc for binding to the nerve growth factor receptor TrkA. A model for discriminating proliferation and differentiation. J Biol Chem 274: 9861–9870. 3. Rabin SJ, Cleghon V, Kaplan DR (1993) SNT, a differentiation-specific target of neurotrophic factor-induced tyrosine kinase activity in neurons and PC12 cells. Mol Cell Biol 13: 2203–2213. PLoS ONE | www.plosone.org 11 January 2012 | Volume 7 | Issue 1 | e29739