Journal: Cell

Article Title: The E3 ubiquitin ligase itch couples JNK activation to TNFalpha-induced cell death by inducing c-FLIP(L) turnover.

doi: 10.1016/j.cell.2006.01.021

Figure Lengend Snippet: Figure 3. Proteasome Activity Is Required for TNFa-Induced c-FLIP Degradation and Cell Death (A) Primary wt hepatocytes were incubated with or without the proteasome inhibitor MG-132 30 min prior to addition of TNFa (10 ng/ml) and CHX (100 mg/ml). At the indicated times (hr), cell lysates were prepared and analyzed by immunoblotting with antibodies for c-FLIPL, IkBa, caspase-3, and actin. (B) shows c-FLIP ubiquitination in livers of ConA-injected mice. wt and Jnk1/ mice were given either vehicle (PBS) or 15.7 ng of a D-JNKi peptide in PBS i.c.v. 30 min before injection of ConA (25 mg/kg). Livers were isolated at the indicated times (hr), lysed, and immunoprecipitated with anti-c-FLIP antibodies, gel separated, and immunoblotted with anti-ubiquitin antibodies. (C) shows Kaplan-Meier survival plot for IkkbDhep mice given PBS or the proteasome inhibitor bortezomib (1 mg/kg) 1 day prior to injection of ConA (25 mg/kg). (D) Ikkb/ fibroblasts were infected with either GFP- or p43-c-FLIPL-expressing lentiviruses and incubated with TNFa (10 ng/ml). After 8 hr, the cells were examined by either PI staining and fluorescent microscopy or bright-field (Br) microscopy.

Article Snippet: We used polyclonal antibodies against caspase-8 (Cell Signaling), c-FLIP (Stressgene), RIP1 (Cell Signaling), p21 (Santa Cruz Biotech), and Bid (Gift from Dr. J. Reed) and monoclonal antibodies against HA (Roche), Myc, His (Invitrogen), c-FLIP (Alexis), actin (Sigma), FADD (Stressgene), ubiquitin, IkBa (Imgenex) JNK1, caspase-3, and cytochrome c (BD biosciences).

Techniques: Activity Assay, Incubation, Western Blot, Ubiquitin Proteomics, Injection, Isolation, Immunoprecipitation, Infection, Expressing, Staining, Microscopy

Journal: Cell

Article Title: The E3 ubiquitin ligase itch couples JNK activation to TNFalpha-induced cell death by inducing c-FLIP(L) turnover.

doi: 10.1016/j.cell.2006.01.021

Figure Lengend Snippet: Figure 4. Itch Is Required for c-FLIP Degradation and TNFa-Induced Apoptosis (A) wt and Itch/ fibroblasts were incubated with TNFa (10 ng/ml) and CHX (100 mg/ml). After 8 hr, apoptotic cells were visualized by PI staining. (B) c-FLIP protein levels and caspase-8 and -3 cleavage were analyzed in wt and Itch/ fibroblasts after TNFa plus CHX treatment. (C) wt, Itchy (Itch/), and Jnk1/ fibroblasts were transduced with an adenovirus encoding IkBa superrepressor (IkBaSR) to inhibit NF-kB. TNFa (10 ng/ ml) was added and at the indicated times cell lysates were prepared and c-FLIPL degradation was examined as above. The extent of apoptosis in parallel treated cultures was examined by propidium iodide staining, and relative rates of apoptotic cell death are indicated below. (D) Itchy fibroblasts were transduced with lentiviruses encoding GFP, wt Itch, or AA-Itch, a mutant in which the D domain required for JNK1 binding and Itch phosphorylation was inactivated (Gao et al., 2004). After 24 hr, the cells were incubated with TNFa plus CHX and Itch expression and c-FLIP degradation was assessed by immunoblotting. Note that the titre of the AA-Itch virus was somewhat higher than that of the wt-Itch virus, and the cells at the 0 time point received more virus. (E) Itch induces c-FLIPL ubiquitination. 293T cells were transfected with Myc-tagged-c-FLIPL, Xpress-tagged Itch, HA-ubiquitin with or without JNKK2-JNK1 (JNK*) expression plasmids. cIAP1 and cIAP2 expression vectors were used as controls for the Itch expression vector. After 36 hr, cells were incubated with TNFa plus CHX for 8 hr and c-FLIPL ubiquitination was examined by immunoprecipitation with anti-Myc antibodies and immunoblotting with anti-HA antibodies. Expression of Itch (not shown) was detected with anti-Xpress antibodies. (F) TNFa stimulates c-FLIPL ubiquitination. 293T cells were transiently transfected with plasmids encoding Myc-tagged c-FLIPL, ubiquitin, and Itch with or without (Mock) a JNK1 vector. The cells were preincubated with MG-132 for 30 min and stimulated with TNFa for the indicated duration (min). After immu- noprecipitation with anti-Myc, ubiquitination of c-FLIPL was examined by immunoblotting as described above. Itch levels were examined by immunoblotting.

Article Snippet: We used polyclonal antibodies against caspase-8 (Cell Signaling), c-FLIP (Stressgene), RIP1 (Cell Signaling), p21 (Santa Cruz Biotech), and Bid (Gift from Dr. J. Reed) and monoclonal antibodies against HA (Roche), Myc, His (Invitrogen), c-FLIP (Alexis), actin (Sigma), FADD (Stressgene), ubiquitin, IkBa (Imgenex) JNK1, caspase-3, and cytochrome c (BD biosciences).

Techniques: Incubation, Staining, Transduction, Mutagenesis, Binding Assay, Phospho-proteomics, Expressing, Western Blot, Virus, Ubiquitin Proteomics, Transfection, Plasmid Preparation, Immunoprecipitation