FIGURE 7.

Proteasome inhibition induces accumulation of K48-linked polyubiquitinated RIPK3. A–F, after induction of RIPK3 with DOX for 14 h, cells were treated with 2 μm MG132 for the indicated time (A) or for 2.5 h (B–F). G, Jurkat cells were treated with 0.5 μm bortezomib for 7 h. Cell lysates were subjected to denaturing IP as described under “Experimental Procedures” (A–G). H–J, after induction of RIPK3 expression by DOX for 14 h, DOX was removed, and the cells were treated with 2 μg/ml cycloheximide for the indicated time. Cycloheximide was removed after 24 h in I due to its toxicity. In J, Ripk3^−/− 3T3 cells expressing tetra-alanine RHIM RIPK3 mutant were used since wild type or kinase dead mutant RIPK3-expressing cells were killed by MG132 much earlier than 24 h (Fig. 2A).