Fig. 5
From: Spermidine reduces ISGylation and enhances ISG15–USP18 interaction
D103A or E133L mutations in mISG15 enhanced ISGylation in the presence of SPD. (A) UBE1L, Ube2l6, HA-Herc5, and FLAG-mISG15 (wild type [WT] or mutant) are expressed in the HEK293T cells in the presence of SPD. 2 days after transfection, the cell lysates are subjected to immunoblotting using anti-FLAG, anti-UBE1L, anti-Ube2l6, and anti-HA antibodies. Ponceau S staining is used as a loading control. Representative data of three independent experiments. (B) Quantification of FLAG-mISGylation, and UBE1L, Ube2l6, and HA-Herc5 expression levels in (A). Each signal is normalized to that of Ponceau S staining. The signal intensity of cells expressing FLAG-mISG15 (WT) and ISGylation system is set as 1. Data are expressed as the mean ± standard deviation of three independent experiments. The means of different groups are compared using the Student’s t-test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively.
