Fig. 1.

Knockdown of COP1 ubiquitin E3 ligase prevents degradation of c-Jun, ETV4, and ETV5 in cells treated with anthrax LT or U0126. (A) Hepa1c1c7 cells were transfected with control (Ctrl) siRNA or siRNA targeting CUL4, COP1, MEKK1, FBW7, SAG, or ITCH ubiquitin E3 ligases. Two days following transfection, the cells were cultured with or without LT for 2 h (Upper) or U0126 for 1 h (Lower) and then extracted with NuPAGE LDS sample buffer. The expression levels of c-Jun protein in the cell lysates were assessed by Western blotting. Reductions in c-Jun levels were statistically analyzed using data from three independent experiments using an unpaired, two-tailed t test and presented as means ± SE (Bottom). Asterisks depict statistically significant differences between the two groups (P < 0.05). (B and C) Hepa1c1c7 cells (B) and Hepa1c1c7 cells stably expressing HA-ETV4 (C, Left) or HA-ETV5 (C, Right) were transfected with control or COP1 siRNA. Two days following transfection, the cells were cultured with medium alone (M) or LT for 2 h or U0126 (U) for 1 h and then extracted with NuPAGE LDS sample buffer. The expression levels of endogenous ETV-4 (B), HA-ETV4 and HA-ETV5 (C) proteins in the cell lysates were assessed by Western blotting. β-Actin levels were used as loading controls. Band intensities were quantified and normalized using β-actin levels. The normalized amounts are shown under each band. Data shown are representative of at least two independent experiments.