SSB cooperates with METTL16-mediated m6A RNA methylation to promote chemoresistance in colorectal cancer cells

Cells grown in 10 cm dishes at 80-90% confluency were lysed with 500 μL NP-40 buffer (150 mM NaCl, 0.5% NP-40, 10 mM Tris-HCl at pH 7.5, and 0.5 mM EDTA containing a protease inhibitor cocktail), followed by sonication. To identify the proteins in the anti-FLAG (METTL16) antibody-immunoprecipitated samples, Nano-liquid chromatography-nanospray tandem mass spectrometry (Nano-LC/MS/MS) of protein identification was performed by the Mass Spectrometry and Proteomics Facility at the Ohio State University (Columbus, Ohio), using a Thermo Scientific orbitrap Fusion mass spectrometer equipped with an nanospray FAIMS Pro™ Sources operated in positive ion mode. Data were searched using Mascot Daemon (Matrix Science, version 2.7.0; Boston, MA) via ProteomeDiscoverer (version 2.4; Thermo Scientific) and the database searched against the most recent Uniprot databases. Label free quantitation was performed using the spectral count approach, in which the relative protein quantitation was measured by comparing the number of MS/MS spectra identified from the same protein in each of the multiple LC/MSMS datasets. Scaffold was used for data analysis.