Fig. 2: Proteosomal degradation of FLIP proteins and inhibition of global protein synthesis upon ER stress in tumor cells.
A HCT116 cells were treated with or without MG-132 (20 μM) for 2 h and then TG (100 nM) was added or not for a further 2-h period. cFLIP levels were determined in whole-cell extracts by western blotting. Levels of both cFLIP isoforms were quantified using GAPDH as protein-loading control through Image LabTM 6.0 software. Blots are representative of three independent experiments. B To analyze cFLIP stability under ER stress, HCT116 cells were treated or not with TG (100 nM) for 2 h prior to treatment with cycloheximide (CHX) (5 μg/mL) for the indicated times. cFLIP levels were determined in whole-cell extracts by western blotting. Levels of cFLIPL were quantified using GAPDH as protein-loading control through Image LabTM 6.0 software and referred to time 0 h levels. Blots are representative of two independent experiments. A longer exposure time is also included (*) to determine cFLIPs stability. C HCT116 cells were treated with or without TG (100 nM) for the indicated times and puromycin (1 μg/mL) was added for the last 10 min of treatment. Puromycin incorporation to the nascent protein chain was assessed by western blotting using an anti-puromycin antibody as described under Materials and Methods. As controls of efficiency of puromycin detection in testing protein synthesis, puromycin was or not added or added in the last 10 min of a 1h-CHX treatment. Ponceau staining was used to test the same amount of protein was present in each lane. Blots are representative of three independent experiments.
