Identified USP5-interacting proteins by mass spectrometry

Empty vector or Flag-USP5 plasmids were transfected into 293T cells. Twenty-four hours later, cells were lysed with IP lysis (25 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% Triton X-100; 10% glycerol) supplemented with protease and phosphatase inhibitors, and total protein was prepared for co-immunoprecipitation with the anti-Flag magnetic beads. Then, immunoprecipitated proteins were prepared for LC-MS/MS according to the manufacturer’s instructions (ProtTech Inc., Suzhou, China). The attachment contains the list of all identified proteins from each sample. Since our results are based on MS/MS peptide sequencing of each individual peptide, all proteins listed here are actual proteins present in a sample instead of “potential candidates”, which are often seen in a MALDI-TOF based peptide mapping. For any identified protein reported here, there should be >98% certainty if the identification is based on LC-MS/MS sequencing of one peptide, and it will have >99.9% certainty if it is based on sequencing of two or more peptides.