Lsd1 antibody

Transfected cells were lysed on ice in RIPA lysis buffer (Thermo Scientific, Waltham, MA, USA) containing 1 × complete protease inhibitor (Roche, Indianapolis, IN, USA). Debris was pelletized by centrifugation at 13 200 r.p.m. for 15 min, and protein concentrations were determined using the Pierce BCA assay (Thermo Scientific). Lysates were heat-denatured at 100 °C for 10 min before separation in 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membrane (GE Healthcare, Mickleton, NJ, USA). Membranes were blocked with 5% bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline Tween-20 buffer (10 mM Tris, pH 7.6, 150 mM NaCl and 0.1% Tween-20) and probed with primary antibody in Tris-buffered saline Tween-20 with 5% bovine serum albumin at the recommended dilutions at 4 °C. Primary antibodies included GPRC5A antibody (Sigma-Aldrich), beta-actin antibody (Cell Signaling Technology, Beverly, MA, USA), HuR antibody (Santa Cruz Biotechnology, Dallas, TX, USA) and LSD1 antibody (Cell Signaling Technology). Membranes were incubated with secondary antibody (Cell Signaling Technology) diluted in Tris-buffered saline Tween-20 with 5% bovine serum albumin for 1 gemcitabine at room temperature. The signal was detected with Pierce ECL Western Blotting Substrate (Thermo Scientific) and GE ImageQuant LAS 4000 (GE Healthcare).

Nuclear proteins were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, 78833), and lysates were pre-cleared with A/G agarose beads (Millipore, LSKMAGA02) for 1 h at 4 °C. LSD1 antibody (Cell Signaling 2139) at 1:50 dilution and IgG Rabbit (Santa Cruz, 2027) at 1:500 dilution were added to each sample and incubated on a rotator over night at 4 °C. The pulldown of LSD1 antibody and IgG control was achieved by adding A/G agarose beads to the samples and incubating them on a rotator for 3 h at 4 °C. Samples were then used for western blot analysis using the following antibodies: LSD1 (Cell Signaling, 2139) and VDR (Santa Cruz Sc-13133 (Clone D-6)).

Whole protein extracts (250 ゼg) in NP40 buffer (150 mM NaCl, 20 mM Tris pH 8.0, 1 mM DTT, 0.5% NP40) were incubated with 15 ゼl of protein A dynabeads (Invitrogen) coupled to 2 ゼg of LSD1 antibody (Cell Signaling) for 3 h at 4°C. After magnetic immunoprecipitation and washes, immunoprecipitates were resolved in a 10% polyacrylamide SDS-PAGE gel, transferred and analyzed by Western blot as described above.