Date Published: April 11, 2019
Publisher: Public Library of Science
Protein lysates, derived from cells following treatment, were obtained by homogenization in RIPA lyses buffer [0.1% sodium dodecylsulfate (SDS), 0,5% deoxycholate, 1% Nonidet, 100 mmol/L NaCl, 10 mmol/L Tris–HCl (pH 7.4), 0.5 mmol/L dithiotritol, and 0.5% phenylmethyl sulfonyl fluoride, protease inhibitor cocktail (Hoffmann-La Roche)] and clarification by centrifugation at 14,000 rpm for 15 min at +4°C. Protein samples containing comparable amounts of proteins, estimated by a modified Bradford assay (Bio-Rad), were subjected to Western blot and immunocomplexes were detected with the enhanced chemiluminescence kit ECL plus, by Thermo Fisher Scientific (Rockford, IL). Membrane pictures were taken by a ChemiDoc System (Bio-Rad, Hercules CA) and bands densities were analyzed with Image Lab Software (Bio-Rad). Equal aliquots of the same sample preparation were loaded and different antibodies were used to reprobe the same blot.