Recombinant ING1 Protein Purification

We transformed ECOS competent Escherichia coli JM-109 cells (Nippon Gene) with the recombinant plasmid pGEX-4 T-1-ING1 and cultured them for 3 h in 200-ml Luria broth containing 0.1 mM isopropyl β-D-thiogalactopyranoside (FUJIFILM Wako Pure Chemical, Osaka, Japan). We then harvested the cells, washed them with phosphate-buffered saline, and lysed them by sonication in Y-PER Yeast Protein Extraction Reagent (Thermo Fisher Scientific). We centrifuged the lysates at 15,000 g for 10 min at 4 °C and purified the GST-fused ING1 proteins in supernatants using affinity chromatography with Glutathione-Sepharose columns (GE Healthcare Life Sciences) according to the manufacturer's instructions. We finally concentrated the purified proteins with an Amicon Ultra-15 Centrifugal Filter Device (Merck KGaA, Millipore, Darmstadt, Germany) as described [12 (link), 13 (link)].