Fig. 5

Modulation of eIF5A1 SUMO2 modification upon stress. A HEK-293 cells were transfected with a plasmid encoding HA-tagged eIF5A1–WT together with pcDNA or Ubc9 and His6–SUMO2. Thirty-six hours after transfection, cells were treated with DMSO or MG132 for 16 h and whole-protein extracts (WCE) and Histidine (His)-tagged purified proteins were analyzed by western blot with the indicated antibodies. B HEK-293 cells were transfected with HA–eIF5A1–WT together with pcDNA or Ubc9 and His6–SUMO2. Thirty-six hours after transfection, cells were subjected to UV irradiation (20 J/m²) followed by 2 h or 6 h rest, and then whole-protein extracts and Histidine-tagged purified proteins were analyzed by western blot with the indicated antibodies. Arrow indicates eIF5A–SUMO2 protein. C HEK-293 cells were transfected with a plasmid encoding HA-tagged eIF5A1–WT together with pcDNA or Ubc9 and His6–SUMO2. Thirty-six hours after transfection, cells were incubated in hypoxic conditions (1% O₂) for 8 h or treated with adriamycin 1 μM for 4 h and then whole-protein extracts and Histidine-tagged purified proteins were analyzed by western blot with the indicated antibodies. Arrow indicates eIF5A1–SUMO2 protein. D HEK-293 cells were transfected with a plasmid encoding HA-tagged eIF5A1–WT together with pcDNA or Ubc9 and His6–SUMO2. Thirty-six hours after transfection, cells were incubated at 43 °C for 2 h and then whole-protein extracts and Histidine-tagged purified proteins were analyzed by western blot with the indicated antibodies. E HEK-293 cells were transfected with a plasmid encoding HA-tagged eIF5A1–WT together with pcDNA or Ubc9 and His6–SUMO2. Thirty-six hours after transfection, cells were first incubated at 43 °C for 2 h (HS) and then incubated at 37 °C for 0.5 or 2 h (hpt HS), as indicated. Then, whole-protein extracts and Histidine-tagged purified proteins were analyzed by western blot with the indicated antibodies. Arrowheads in all panels indicate unconjugated eIF5A1 protein