Supplementary Materials and Methods
Immunofluorecense
Cells were stained according to Dallol et al., 2004. In brief cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After fixation, cells were permeabilized with 0.1% Triton X-100 in PBS/1% BSA for 2 min. Nonspecific binding was blocked by incubating the coverslips with PBS/1% BSA for 30 min at room temperature. Incubation with the indicated antibody was done at room temperature for 1 h followed by extensive washes in PBS. This was followed by incubation for 30 min at room temperature with the appropriate secondary antibody and several washes in PBS. The coverslips were mounted on glass slides using Vectashield with 4',6-diamidino-2-phenylindole mounting medium. Images were visualized with a Photometrics SenSys KAF 1400-G2 charged coupled device fitted to a Zeiss Axioplan fluorescence microscope and captured using the SmartCapture software from Digital Scientific.
Identification of novel interacting partners using MS
Immunoprecipitation and identification using 2D-LC/MS/MS was done as described (von Kriegsheim et al., 2006) with minor modifications. MCF7 cells (2x108) stably expressing FLAG-RASSF2 were lysed in 10ml of buffer containing 20mM Hepes (pH 7.5), 150 mM NaCl, 2 mM EDTA, 0.5% NP-40, 1 mM PMSF, 2 mM NaF, 1 mM Na vanadate, 5 μg/mL leupeptin and 2.2 μg/mL aprotinin, 1 mM Sodium pyrophosphate 20 mM ß-Glycerophosphate. After incubation on ice for 10 min, the lysates were clarified by centrifugation at 4000 rpm for 15 mins at 4°C. Meanwhile 100 μL of anti-FLAG M2 affinity gel (Sigma-Aldrich) was washed in lysis buffer, then incubated with the lysate for 2h. The beads were pelleted by centrifugation and washed 3 times with lysis buffer with 50mM NaCl, 0.1% NP-40 without sodium pyrophosphate. The proteins were then eluted from the beads by two 5 min incubations with FLAG-peptide (150ug/ml from Sigma-Aldrich). The eluate was concentrated (Eppendorf Microcon Ultracel YM-3) then resolved through a Novex NuPAGE 10% Bis-Tris 1mm gels (Invitrogen). After colloidal staining (Sigma-Aldrich B-2025 Brilliant blue), the gel lane was divided into 12 portions and each subject to in gel trypsin digestion. Tryptic peptides were separated by reverse phase chromatography and analysed online by a Q-STAR (Applied Biosystems) mass spectrometer (described in detail in (Kriegsheim et al., 2008)). Peptides fragmentation patterns were searched against Swiss-Prot and NCBI databases using Mascot (Matrix Science).