Proteome Extraction and Quantification

Cell samples were collected and processed as described for proteome extraction, quantization and digestion [45 (link),46 (link),47 (link)]. Briefly, six biological cell replicates per condition were lysed in RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) applying mechanical homogenization with TissueLyser II homogenizer (Qiagen, Duesseldorf, Germany). Lysates were treated with 1% Benzonase (E8263-5KU, Sigma-Aldrich, St. Louis, MO, USA) in 2 mM MgCl₂ at 37 °C for 30 min, and then centrifuged at 18,000 rpm for 30 min at 4 °C to discard membranes, cell debris and nucleic acids. Digestion of proteomes was performed at 47 °C for four hours on S-Trap^TM micro spin columns (Protifi, Huntington, NY, USA), using trypsin (Promega, Madison, WI, USA) 1:25 per 50 µg of protein extracts. Eluted peptides were injected onto a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system, constituted by an EASY-nLC^TM coupled with a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific, Bremen, Germany) [48 (link)].