To identify in vivo methylation sites of Plk1, 293T cells were transfected with Flag-tagged Plk1. After 36 hours, the cells were harvested and lysated. Flag-Plk1 was immunoprecipitated by α-Flag resin and subjected to SDS-PAGE. The band corresponding to Plk1 was subjected to in-gel trypsin digestion and desalted with C18 tips. The samples were analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) on a Q Exactive-HF mass spectrometer (Thermo Fisher Scientific). The LC-MS/MS data were processed using Proteome Discoverer (Thermo Fisher Scientific) and searched against the Swiss-prot Homo sapiens protein sequence database. The existence of monomethylation was verified on the basis of the occurrence of consecutive y or b ions and the mass increment of 14.016 Da on lysine residues.

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