FIGURE 3.

Phosphorylation of Thr-166 and ubiquitination of Lys-167 are required for ROS-dependent ubiquitination and degradation of FLIP following menadione treatment. A, PPC1 cells were transfected with pcDNA3.1-His₆ vector (first lane) or various His-tagged FLIP plasmids (WT, T166A, K167R, and T166A,K167R double mutant) for 16 h. Cells were then treated with menadione (5 μm) in the presence of MG132 (1 μm) for 8 h. His-tagged FLIP proteins were captured by Ni-NTA resin and eluted by imidazole solution. The Ni-NTA-purified proteins were analyzed by immunoblotting using mouse anti-FLIP and anti-Ubiquitin antibodies. The inputs (1/20 of lysates used for FLIP protein capture) were analyzed by immunoblotting using rabbit anti-FADD as a loading control. B, levels of His-FLIP protein in A were quantified using scanning densitometry. Vector groups with no treatment were adjusted to 1. Statistical significance (mean ± S.E.; n = 3) was determined by two-way analysis of variance and Bonferroni post-test. *, indicates the p value is p < 0.05; **, indicates p < 0.001.