Protein digestion and mass spectrometry analysis were executed as previously described 24. In brief, HT29 cells treated with azelastine for 48 h were lysed with a lysis buffer. Proteins were digested with trypsin, vacuum-freeze-dried, and resuspended in anhydrous acetonitrile solution, then desalted with MonoTIPTM C18 Pipette Tip (GL Sciences, Tokyo, Japan). Peptide samples were analyzed with an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Then raw data were analyzed using Proteome Discoverer (Thermo Fisher Scientific) and Spectronaut (Omicsolution Co., Ltd., Shanghai, China) software. Protein and peptide FDRs were set to 1%, and Ingenuity Pathway Analysis (IPA, Ingenuity Systems, Redwood City, CA) was used to analyze the differentially expressed proteins.

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