A site localization probability of at least 0.75 was used as the threshold for confident localization (Vizcano em et?al /em , 2013, 2016). Cell proliferation assay Human being adenoid main B cells were stained with CellTrace Violet according to the manufacturer’s instructions (Thermo Fisher Scientific). windowpane was set to 1 1.7 Th, and MS/MS spectra were acquired in the Orbitrap with a resolution of 15,000 at Diosmetin-7-O-beta-D-glucopyranoside 200, using an AGC target value of 2e5, and a maxIT of 75?ms. Dynamic exclusion was arranged to 20?s. Peptide and protein recognition was performed using MaxQuant (version 1.5.3.30) with its built in search engine Andromeda (Cox & Mann, 2008). Spectra were looked against a SwissProt Diosmetin-7-O-beta-D-glucopyranoside database, either the (OX 7108 \ 26,502 sequences) or (UP000002032 C 4,156 sequences), supplemented with the EBNA2 protein sequence. Enzyme specificity was arranged to Trypsin/P or GluC accordingly, and the search included cysteine carbamidomethylation as a fixed modification, protein N\term acetylation, oxidation of methionine, and phosphorylation of serine, threonine, tyrosine residue (STY) as variable modifications. Up to two and three missed cleavage sites were allowed for trypsin and GluC, respectively. Precursor tolerance was arranged to 4.5?ppm (after MS1 feature re\calibration) and fragment ion tolerance to 20?ppm. The match between runs feature was enabled. Peptide recognition was further filtered for a minimum Andromeda score of 20 or 40, for unmodified and revised (phosphorylated) sequences, respectively. A site localization probability of at least 0.75 was used SEL-10 as the threshold for confident localization Diosmetin-7-O-beta-D-glucopyranoside (Vizcano em et?al /em , 2013, 2016). Cell proliferation assay Human being adenoid main B cells were stained with CellTrace Violet according to the manufacturer’s instructions (Thermo Fisher Scientific). Proliferation of CD19+ B cells was monitored by circulation cytometry using BD Fortessa, and the data were analyzed using the FlowJo software (Version 10.5.3). Dual\luciferase assay 5??106 DG75 cells were electroporated with 1.5?g EBNA2 expression plasmids and the luciferase construct 1.5?g pGa981.6 (Minoguchi em et?al /em , 1997) carrying a multimerized CBF1 binding site to measure EBNA2 activity and 0.2?g Renilla luciferase expression plasmid. 24?h post electroporation, cells were washed, pelleted, and lysed in 100?l lysis buffer for 30?min on snow. Cell extracts were tested from the dual\luciferase assay according to the manufacturers instructions (Promega). Author contributions XZ, MS, CM, RK, and BKe conceptualized the study; AMo, PG, and SMH involved in formal analysis and data curation; XZ, PS, MR, KS, and CM contributed to methodology; BKe and CM involved in funding acquisition; XZ, PS, AMo, PG, AMu, ST, CK\R, SB, and RK investigated the study; WH, MR, MS, CM, KS, BKu, and CM offered resources; WH, RK, MS, CM, and BKe supervised the study; XZ, PS, AMo, and BKe visualized the study; XZ and BKe wroteoriginal draft; XZ, MS, CM, PG, PS, AMo, BKe, and RK wrotereview & editing. Discord of interest The authors declare that they have no discord of interest. Supporting information Expanded View Numbers PDF Click here for more data file.(1.8M, pdf) Table?EV1 Click here for more data file.(19K, docx) Table?EV2 Click here for more data file.(16K, xlsx) Resource Data for Number?1 Click here for more data file.(15M, pdf) Resource Data for Number?2 Click here for more data file.(43M, pdf) Resource Data for Number?3 Click here for more data file.(7.7M, tif) Resource Data for Number?4 Click here for more data file.(12M, pdf) Resource Data for Number?5 Click here for more data file.(126K, pdf) Acknowledgements We thank Dagmar Pich, Yen\Fu Adam Chen, and Ezgi Akidil for all the excellent suggestions they gave. We say thanks to Kerstin Heise, Michaela Kroetz\Fahning, and Andreas Klaus for expert technical assistance. This project was supported from the Wilhelm Sander\Stiftung (Give 2015.165.1) to Bettina Kempkes. Xiang Zhang is definitely supported from the China Scholarship Council (CSC No.: 201603250052). Cristian Mnz is definitely financially supported by Cancer Study Switzerland (KFS\4091\02\2017 and KFS\4962\02\2020), Swiss National Science Basis (310030B_182827, 310030L_197952/1, and CRSII5_180323). Open Access funding enabled and structured by Projekt DEAL. Notes EMBO reports (2021) 22: e53007. [PMC free article] [PubMed] [Google Scholar] Contributor Info Michael Sattler, Email: ed.nehcneum-ztlohmleh@relttas. Christian Mnz, Email: hc.hzu.ygolonummi@czneum. Bettina Kempkes, Email: ed.nehcneum-ztlohmleh@sekpmek. Data availability Phosphorylation of EBNA2 by PLK1: The mass spectrometry proteomic data have been deposited in the ProteomeXchange Consortium via the PRIDE partner.