Subcellular protein fractionation kit for culture cells

HEK-293 cells were rinsed with phosphate-buffered saline (PBS) and lysed with lysis buffer (10 mM HEPES, pH 8.0, 40 mM KCl, 3 mM MgCl₂, 5% glycerol, 2 mM DTT, 0.5% Nonidet P-40) for 10 min at 4°C. Whole cell lysates were prepared by performing centrifugation (4250 g for 5 min at 4°C), and the supernatant was collected and stored at −80°C. For cytoplasmic and nuclear extracts preparation, cells were fractionated using Subcellular Protein Fractionation Kit for Culture Cells (Thermo Scientific) according to the manufacturer's instructions. Briefly, cells were harvested with trypsin-ethylenediaminetetraacetic acid and then washed with ice-cold PBS. The cytoplasmic and nuclear extracts were extracted by incubating cells with cytoplasmic extraction buffer or nuclear extraction buffer, respectively.

Chromatin was extracted using the Subcellular Protein Fractionation Kit for Culture Cells (Thermo Fisher Scientific, 78840) according to the manufactures instructions with the exception that the provided protease inhibitors were substituted with 0.5 mM pefablock (Sigma-Aldrich, 11429868001). Equal amounts of chromatin (30 µg protein) were incubated at 37 °C for 15 min in a pH-modified cathepsin reaction buffer (50 mM sodium acetate, 4 mM EDTA, pH 7.0), containing 0.5 mM pefabloc, 5 mM dithiothreitol (Sigma-Aldrich, D9779), and indicated amounts of rCTSB (Biovison, 7580), rCTSL (Biovison, 1135) and ALLN (Calbiochem, 208719). Samples were subsequently boiled for 5 min and analyzed by immunoblotting.