Anti pfk 1 h 55

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.

Cells were washed in ice-cold PBS, lysed using RIPA buffer with protease inhibitors (Complete, Roche Diagnostics) and then centrifuged (250 × g, 5 min) to remove the debris. After measuring protein concentration by Bradford assay, samples containing 20 μg protein were mixed with 5 × loading buffer, denatured for 2 min at 95°C, loaded into a 12% polyacrylamide gel, run at 100 V for 2 h and then transferred onto a PVDF membrane (Millipore) for 3 h at 40 V. Next, the membrane was blocked with 5% non-fat dried milk powder (Applichem) in Tris-buffered saline (TBS)/ 0.1% Tween 20 (TBST) and then incubated with anti rabbit-PFKFB3 (Sigma), anti-beta-actin mAb (Abcam), anti-PFK-1 (H-55) (Santa Cruz Biotechnology) overnight at 4°C. The membrane was then washed 3 × 10 min with TBST and incubated with appropriate horseradish peroxidase–conjugated secondary antibodies for 1 h. The membrane was further washed 3 × 10 min with TBST before development using Amersham ECL Plus Western blotting developing kit (GE Technologies). Bands on membranes were visualized with a luminescence image analyzer (LAS 1000, Fuji Film) and analyzed with Image J software (available at http://rsb.info.nih.gov/ij; developed by Wayne Rasband, NIH).