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. 2010 Dec 23;286(9):6946–6954. doi: 10.1074/jbc.M110.172338

FIGURE 4.

FIGURE 4.

Phosphorylation of Thr-263 plays a critical role in stabilizing caspase-8. A, phosphorylation of caspase-8 at Thr-263 enhances ubiquitination of caspase-8. Caspase-8-WT and mutant caspase-8-T263A were each transfected with HA-Ub with or without RSK2 and cultured for 30 h. The cells were treated with MG132 for 4 h, and then proteins were extracted. The caspase-8 proteins were immunoprecipitated with anti-V5, and ubiquitinated caspase-8 proteins were visualized by Western blot with anti-HA. B, phosphorylation of Thr-263 plays an important role in caspase-8 stability. Caspase-8 or mutant caspase-8-T263A was transfected into HeLa cells. Cells were cultured for 30 h and then treated with EGF (100 ng/ml) plus CHX (30 μg/ml) for the indicated time, and then proteins were extracted. The caspase-8 protein abundance was visualized by Western blot with anti-V5 antibody. The graph shows data from triplicate experiments (mean ± S.D.). The asterisk (*) indicates a significant difference (p < 0.05, Student's t test). C, MG132 rescues caspase-8 expression. Caspase-8 was transfected into HeLa cells, and cells were cultured for 30 h. Cells were then pretreated with MG132 (20 μm) for 1 h followed by treatment with EGF (100 ng/ml) plus CHX (30 μg/ml) for 6 h. The proteins were extracted, and caspase-8 protein abundance was visualized by Western blot with anti-V5. D, Thr-263 is critical for Fas induction of apoptosis. GFP-caspase-8-WT or GFP-caspase-8-T263A was transfected into Jurkat I9.2 cells, and cells were cultured for 24 h. Cells were exposed to anti-Fas (200 ng/ml) for 6 h and then either subjected to immunoblotting to detect GFP, caspase-8, caspase-3, or β-actin (top), or apoptosis was analyzed by annexin V and propidium iodide staining using flow cytometry (bottom). Data are represented as means ± S.D. of the percentage of annexin V-positive cells determined from triplicate experiments. The asterisk (*) indicates a significant difference (p < 0.05, Student's t test; bottom). IB, immunoblot; cleav, cleaved; endo, endogenous.