Immunoprecipitated STAT3 from total liver lysates or BMMs was subjected to SDS-PAGE. The bands corresponding to STAT3 were excised, and the proteins were reduced, alkylated, and in-gel digested with trypsin. The peptides were extracted, lyophilized, resuspended in 2%ACN/0.1% formic acid, and analyzed by LC-MS/MS using a nanoAcquity (Waters Corp.) coupled to an LTQ Orbitrap Velos (Thermo Fisher Scientific). Samples were injected onto an in-house packed C18AQ (Michrom Biosciences) trap column (100 µm internal diameter × 2 cm, 5 µm particle size) and were washed with 1% ACN/0.1% formic acid for 20 min at 2 µl/min, then loaded onto an analytical column (C18AQ, 100 µm internal diameter × 22 cm, 5 µm particle size). The linear gradient for separation consisted of 5–36% mobile phase B over 120 min at a 250-nl/min flow rate, where mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in ACN. The LTQ Orbitrap Velos was operated in data-dependent mode: the 10 most intense precursors were selected for subsequent collision-induced dissociation (CID) fragmentation. Resolution for the precursor scan (m/z 300–2,000) was set to 60,000 at m/z 400 with a target value of 106 ions. The MS/MS scans were acquired in the linear ion trap with a target value of 1,000. The normalized collision energy was set to 35% for CID. Precursors with unknown charge or a charge state of 1 and 4 or higher were excluded. Raw data files were processed using Proteome Discoverer version 2.0 (Thermo Fisher Scientific). Peak lists were searched against a Homo sapiens Uniprot database using Sequest. The following parameters were used to identify tryptic peptides for protein identification: 10 ppm precursor ion mass tolerance; 0.6 D product ion mass tolerance; up to two missed trypsin cleavage sites; carbamidomethylation of Cys set as a fixed modification; and hexNAc (+203.0794 D) of N/S/T, oxidation of M, and phosphorylation of S/T/Y set as variable modifications. The Percolator node was used to determine false discovery rates (FDRs), and a peptide FDR of 5% were used to filter all results.