Fig. 2: K26 residue of YBX1 was one key SUMOylation site.

A YBX1 was modified by the exogenous His-tagging SUMO1^T95K (His-SUMO1^T95K) protein. Under co-overexpression of Flag-YBX1 with His-SUMO1^T95K in HEK293T cells, the Flag-YBX1 was enriched by IP using anti-Flag magnetic beads to detect the SUMOylated YBX1 level. B The K52, K92, K118 and K137 residues of YBX1 were identified as the SUMOylation sites by MS/MS. C The MS/MS spectrum of one peptide (YLRSVGDGETVEFDVVEGEK¹¹⁸GAEAANVTGPGGVPVQGSK) from YBX1 whose K118 was conjugated with SUMO in FTMS/CID fragmentation mode. D The levels of SUMOylated YBX1 were significantly reduced in the four K sites-mutant (4KR) YBX1 with the simultaneous K52R/K92R/K118R/K137R mutation. HEK293T cells were co-transiently transfected with pHA-UBC9, pMyc-SUMO1 and pFlag-YBX1 (or each of pFlag-YBX1^K52R, pFlag-YBX1^K92R, pFlag-YBX1^K118R, pFlag-YBX1^K137R, pFlag-YBX1^4KR) for 48 h, and cell lysates were performed IP to capture Flag-YBX1 to detect the SUMOylated YBX1 level. E The K26 residue of YBX1 was identified being the major SUMO modification site. Top: Diagram for domain organization of YBX1 and the locations of all K residues. Bottom: Different single K-point mutants of YBX1 were transfected into HEK293T cells as indicated to measure YBX1 SUMOylation level. F The SUMOylation level of YBX1 was almost completely abolished in the five K sites-mutant (5KR) of YBX1 with the simultaneous K26/K52R/K92R/K118R/K137R mutation. HEK293T cells were co-transiently transfected with pHA-UBC9, pMyc-SUMO1 and pFlag-YBX1 or each of pFlag-YBX1^K26R, pFlag-YBX1^4KR, pFlag-YBX1^5KR. After 48 h later, cell lysates were extracted to capture Flag-YBX1 and detect the SUMOylated YBX1 level. S-Flag-YBX1 SUMOylated Flag-tagging YBX1, S-YBX1 SUMOylated YBX1, IP immunoprecipitation, IB immunoblot, Input Same account of cell lysate to load.