Fig 1: Downregulation of ISGylation but not of UBE1L, UBE2L6, Herc5, and USP18 via curcumin treatment. (A) Curcumin partly prevents ISGylation in MCF10A cells. MCF10A cells were stimulated by human IFNa with or without curcumin (5 or 10 µM) for 2 d. The cell lysates were subjected to immunoblotting with an anti-ISG15, anti-UBE1L, anti-UBE2L6, anti-Herc5, or anti-USP18 antibody. Ponceau S staining was used as loading control. Representative data of three independent experiments. (B) Quantification of UBE1L, UBE2L6, Herc5, and USP18 expression levels, as well as of ISGylation in (A). Each signal was normalized to that of Ponceau S staining. The signal intensity of cells stimulated by human IFNa was set as 1. Data are expressed as the mean ± standard deviation of three independent experiments. Means of different groups were compared using Student's t-test. A P value < 0.05 was considered statistically significant. (C) Curcumin partly prevents ISGylation in A549 cells. A549 cells were stimulated by human IFNa with or without curcumin (5 or 10 µM) for 2 d. The cell lysates were subjected to immunoblotting with an anti-ISG15, anti-UBE1L, anti-UBE2L6, anti-Herc5, or anti-USP18 antibody. Ponceau S staining was used as loading control. Representative data of three independent experiments. (D) Quantification of UBE1L, UBE2L6, Herc5, and USP18 expression levels, as well as of ISGylation in (C). Each signal was normalized to that of Ponceau S staining. The signal intensity of cells stimulated by human IFNa was set as 1. Data are expressed as the mean ± standard deviation of three independent experiments. IFNa, interferon alpha; ISG15, interferon-stimulated gene, 15 kDa; ISGylation, ISG15 modification; UBE1L, ubiquitin-activating enzyme E1-like protein; UBE2L6, ubiquitin-conjugating enzyme E2 L6; Herc5, probable E3 ubiquitin-protein ligase; USP18, ubiquitin-specific peptidase 18. Means of different groups were compared using Student's t-test. A P value < 0.05 was considered statistically significant.