O 2 acetamido 2 deoxy d glucopyranosylidenamino n phenylcarbamate pugnac

Frozen RV samples were pulverized and incubated in solubilization buffer (0.025M Tris, 0.15M sodium chloride, 0.001M ethylenediaminetetraacetic acid, 1% NP40, 5% glycerol, pH 7.4) supplemented with mammalian protease inhibitor (Sigma-Aldrich) and the OGA inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidenamino) N-phenylcarbamate (PUGNAc, Sigma-Aldrich) (100 µM). Protein concentration was determined using a BCA kit and then 2.5 mg of total protein extract was incubated with the pan O-GlcNAcylation monoclonal antibody RL2 (ThermoScientific) covalently linked to metallic beads (Pierce Co-Immunoprecipitation kit) overnight at 4 °C. The beads were washed with solubilization buffer twice and once with H₂O. Protein was eluted from antibodies using acidic solution. Neutralization buffer was added to solutions prior to proceeding with mass spectrometry analysis. Raw proteomics data is supplied in Supplemental Table S4.

DON, O-(2-Acetamido-2-deoxy-D-glucopyranosylidenamino) N-phenylcarbamate (PUGNAc), and cycloheximide were obtained from Sigma-Aldrich (St. Louis, MO). Antibodies were purchased from various sources: anti-β-catenin (14) and anti-E-caherin (36) from BD biosciences; anti-slug (C19G7) and anti-vimentin (D21H3, for western blot) from Cell Signaling (Danvers, MA); anti-O-GlcNAc (RL-2) from Pierce Biotechnology (IL, USA); anti-GFAT (H-49), anti-HK-II (C-14), anti-OGT (F-12), and anti-PFK-1 (H-55) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-OGA and control IgG for immunoprecipitation from Sigma-Aldrich (St. Louis, MO); anti-vimentin (RV202) for immunoprecipitation from Abcam (Cambridge, UK). Detail of all antibodies are listed in Supplementary Table S1.