Fig. 1

eIF5A1 protein is modified by SUMO2 in vitro and in vivo in an hypusine-independent manner. A In vitro SUMOylation assay with SUMO2 using ³⁵S-methionine labeled in vitro translated HA–eIF5A1 protein. B HEK-293 cells were cotransfected with a plasmid encoding Flag–eIF5A1 and pcDNA or Flag–eIF5A1, Ubc9, and His6–SUMO2. Thirty-six hours after transfection, total protein extracts (WCE) and proteins fused to Histidine (His) tag were analyzed by western blot with the indicated antibodies. C SUMOylation of endogenous eIF5A1. SUMOylated proteins purified from HEK-293 cells using SUBES were analyzed by western blot with anti-eIF5A1 or anti-SUMO2 antibody. D Amino acid sequence homology between human eIF5A1 and eIF5A2. E In vitro SUMOylation assay using ³⁵S-methionine labeled in vitro translated eIF5A2 protein and SUMO2. F HEK-293 cells were co-transfected with Flag–eIF5A2 and pcDNA or Flag–eIF5A2, UBC9, and His6–SUMO2. Thirty-six hours after transfection, total protein extracts and proteins fused to Histidine tag were analyzed by western blot with the indicated antibodies. G In vitro SUMOylation assay in the presence of SUMO2 and using ³⁵S-methionine labeled in vitro translated eIF5A1–WT or the eIF5A1–K50R hypusination mutant as a substrate. H HEK-293 cells were transfected with HA–eIF5A1–WT or HA–eIF5A1–K50R together with pcDNA or Ubc9 and His6–SUMO2. Thirty-six hours after transfection, whole-protein extracts and Histidine-tagged purified proteins were analyzed by western blot with the indicated antibodies (left panel). The ratio of SUMOylated eIF5A to unmodified eIF5A is shown (right panel). Data represent the mean and error bars of three biological replicates. I In vitro translated Flag–eIF5A1–WT protein was subjected to an in vitro SUMOylation assay in the presence of SUMO2 and the SUMOylation products were analyzed by western blot with anti-hypusine or anti-Flag antibodies. Arrowheads in all panels indicate unconjugated eIF5A protein. Arrows indicate the SUMO2-conjugated eIF5A protein