Anti flag conjugated beads

HEK293T or U87 cells were transfected with pCMV-Flag-RhoE (RND3) or pCMV-Flag as a control as previously described [23 (link)]. To discover putative RND3 binding proteins cell lysates were immunoprecipitated using anti-Flag conjugated beads (Sigma). After SDS-PAGE and staining with coomassie blue the gel was cut into 7 equal fragments and subjected to in-gel trypsin digestion (along with matching gel slices from empty vector control) and analysed by mass spectrometry.
Co-immunoprecipitation of Flag-RND3 with Mcm3 was performed on U87 cells. Cells were washed with ice-cold serum-free medium and lysed on ice in buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EGTA (pH 8.0), 1 mM EDTA (pH 8.0), 2.5 mM pyrophosphate, 1 mM β-glycerophosphate, 1% Triton X-100 containing freshly added protease, and phosphatase inhibitor cocktail tablets (Roche). Lysates were clarified by centrifugation at 4°C, and the protein concentrations were determined by using Bio-Rad protein assay reagent (Bio-Rad Laboratories). For immunoprecipitation analyses, aliquots of cellular lysates were incubated with 2 μg of monoclonal anti-Flag (Sigma M2) for 1 h at 4°C. Immunocomplexes were collected on protein G-Sepharose beads (Sigma). The beads were washed three times with lysis buffer then boiled for 5 min in Laemmli sample buffer.

For the methyl-lysine pulldown assay, GST-3xMBT protein (the GST-3xMBT plasmid is a gift from Dr. Or Gozani) was expressed in E. coli DH5α cells and purified by glutathione Sepharose 4B chromatography following the manufacturer's protocol (Amersham Biosciences). Nuclear extracts from VCaP cells treated with or without 1 mmol/L pargyline were used for the pulldown assay with GST-3xMBT protein and the immunopurified proteins were separated by SDS-PAGE gel, stained with Coomassie blue, and then subjected to mass spectrometry analysis. For other immunoprecipitation (IP) assays, cells were lysed in Triton Lysis buffer and treated with protein inhibitor cocktails (Thermo Fisher Scientific), followed by a brief sonication, and then lysates were immunoprecipitated with anti-V5-conjugated beads (Sigma, A7345) or anti-FLAG-conjugated beads (Sigma, A2220). For immunoblotting, proteins were detected with primary antibodies, including anti-V5 (Abcam, ab9116), anti-EHMT1 (Abcam, ab41969), anti-EHMT2 (Novus, NBP2-13948), anti-FLAG (Sigma, F3040), anti-LSD1 (Abcam, ab17721), anti-CoREST (Abcam, ab183711), anti-HDAC1 (Abcam, ab7028), anti-HDAC2 (Abcam, ab7029), anti-REST (Millipore, CS200555), anti-E2F1 (Cell Signaling Technology, 3742), anti-GAPDH (Abcam, ab8245), anti-H3K9me1(Abcam, 9045), anti-H3K9me2 (Cell Signaling, 9753S), and anti-Tubulin (Abcam, ab6046).