Fig. 3

Mutation of the SUMOylation sites in eIF5A1 modulates its stability. A HEK-293 cells were transfected with HA–eIF5A1–WT, HA–eIF5A1–3KR, or HA–eIF5A1–5KR, and treated with cycloheximide (CHX) 24 h after transfection. Samples were collected at the indicated hours post treatment (hpt) and protein extracts were analyzed by western blot with the indicated antibodies (left panel). The intensity of the bands was quantified using ImageJ software. eIF5A band intensities were normalized to GAPDH bands from each respective time and plotted (right panel). Data represent the mean and error bars of three biological replicates. Statistical analysis was assessed by a Student’s t-test. *P < 0.05, **P < 0.01, compared with WT. B HEK-293 cells were transfected and treated as indicated in A but in presence or absence of MG132 treatment. At the indicated times after CHX treatment, protein extracts were analyzed by western blot with the indicated antibodies (upper panels). The intensity of the bands was quantified using ImageJ software. eIF5A band intensities were normalized to actin bands from each respective time and plotted (lower panels). Data represent the mean and error bars of three biological replicates. Statistical analysis was assessed by a Student’s t-test. *P < 0.05, **P < 0.01. C HEK-293 cells were transfected with HA–eIF5A1–WT, HA–eIF5A1–5KR, or HA–eIF5A–3KR, and 24 h after transfection cells were treated with DMSO or ML-792 (500 nM) for 16 h. Then, the cells were treated with cycloheximide (CHX) for the indicated times and protein extracts were analyzed by western blot with anti-HA antibody (upper panels). The intensity of the bands was quantified using ImageJ software. eIF5A band intensities were normalized to GAPDH bands from each respective time and plotted (lower panels). Data represent the mean and error bars of three biological replicates. Statistical analysis was assessed by a Student’s t-test. *P < 0.05