Fig. 2

Identification of the SUMO conjugation sites on eIF5A1. A, B In vitro SUMOylation assay with SUMO2 and the indicated ³⁵S-methionine-labeled in vitro translated eIF5A1 proteins. C HEK-293 cells were transfected with a plasmid encoding HA-tagged eIF5A1–WT, eIF5A1–3KA, or eIF5A1–5KA. Thirty-six hours after transfection, cells were treated with MG132 for 16 h and protein extracts were analyzed by western blot with anti-HA and anti-GAPDH antibodies. D HEK-293 cells were transfected with a plasmid encoding HA-tagged eIF5A1–WT, eIF5A1–3KA, or eIF5A1–5KA together with pcDNA or Ubc9 and His6–SUMO2. Thirty-six hours after transfection, cells were treated with MG132 for 16 h and whole-protein extracts (WCE) and Histidine (His)-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, eIF5A1–3KR, or eIF5A1–5KR together with pcDNA or Ubc9 and His6–SUMO2. Thirty-six hours after transfection, cells were treated with MG132 for 16 h and whole-protein extracts (WCE) and Histidine (His)-tagged purified proteins were analyzed by western blot with the indicated antibodies. Arrowheads in all panels indicate unconjugated eIF5A1 protein. Arrows indicate the SUMO2-conjugated eIF5A1 protein