Fig. 5. Inhibition of GSK3 does not alter c-FLIP mRNA levels (A), rather facilitates ubiquitin/proteasome-mediated c-FLIP degradation (B and C) independent of Itch (D).

A, The indicated cell lines were treated with the given concentrations of SB216763 for 6 h and then subjected to preparation of total cellular RNA and subsequent detection of c-FLIP using RT-PCR. B, The given cell lines were pretreated with 20 µM MG132 for 30 minutes prior to the addition of 20 µM SB216763. After co-treatment for 4 h, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. C, H157-FLIP_L-21 cells which stably express ectopic flag-FLIP_L were transfected with HA-ubiquitin plasmid using FuGENE 6 transfection reagent for 24 h. The cells were then pretreated with 20 µM MG132 for 30 minutes and then co-treated with 20 µM SB216763 for 4 h. Whole-cell protein lysates were then prepared for immunoprecipitation using anti-Flag antibody followed by Western blotting (WB) using anti-HA antibody for detection of ubiquitinated FLIP_L (Ub-FLIP_L) and anti-Flag antibody for detection of ectopic FLIP_L. D, H157 cells were transfected with the indicated siRNAs for 48 h and then treated with 20 µM SB216763 for additional 8 h. The cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis for detection of the indicated proteins.