1. Volume 17, Issue 5, Pages 418-424 (March 2007)
Ubiquitination of RIP1 Regulates an NF-κB-Independent Cell-Death Switch in TNF Signaling 
Marie Anne O'Donnell, Diana Legarda-Addison, Penelopi Skountzos, Wen Chen Yeh, Adrian T. Ting 
Current Biology 
Volume 17, Issue 5, Pages (March 2007)
DOI: /j.cub
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2. Figure 1 Lysine 377 of RIP1 Regulates Cell Death
(A) RIP1-null cells and RIP1-null cells reconstituted with RIP1-WT or RIP1-K377R were stimulated with the indicated doses of TNF for 5 and 24 hr. Cells were stained with annexin V-PE and analyzed by flow cytometry. The percentage of total cells that were reactive to annexin V is shown.
(B) The three cell lines in (A) were stimulated with 10 ng/ml TNF for the indicated times, and annexin-V levels were analyzed.
(C) RIP1-WT and RIP1-K377R cells were stimulated with 10 ng/ml TNF for different periods of time (left panel) or the indicated doses of TNF for 24 hr (right panel). Cell lysates were blotted with antibodies specific for CASPASE-8, cleaved CASPASE-3, or cleaved PARP. The anti-PARP blots were also overexposed to visualize additional PARP cleavage products. Reblotting with anti-tubulin and PLCγ demonstrated equivalent loading. These western blots are representative of at least three similar experiments.
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3. Figure 2
Lysine 377 of RIP1 Provides an Early NF-κB-Independent and a Late NF-κB-Dependent Antiapoptotic Signal
(A) RIP1-null, RIP1-WT, or RIP1-K377R cells were transduced with retroviruses encoding either GST as a negative control (left panels) or IκBαSR (right panels). These cells were stimulated with the indicated doses of TNF for 5 hr (panels I and II) and 24 hr (panels III and IV). Apoptosis was analyzed by annexin V staining and flow cytometry as in Figure 1A.
(B) The cell lines in (A) were stimulated with 10 ng/ml TNF, and apoptosis was analyzed at the indicated times.
(C) RIP1-WT and RIP1-K377R cells stably transfected with the Myc-tagged IκBαSR were stimulated with the indicated doses of TNF for 24 hr (right panel) or 10 ng/ml TNF for different periods of time (left panel). Cell lysates were blotted as in Figure 1C. The anti-Myc blot both serves as a loading control and shows equivalent expression of the IκBαSR in the two cell lines. These western blots are representative of at least three similar experiments.
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4. Figure 3
UBC13 and TRAF2 Have an NF-κB-Independent Antiapoptotic Function
(A) IKKγ/NEMO-deficient Jurkat T cells stably expressing the control protein GST, UBC13DN, or TRAF2DN were treated with the indicated concentrations of TNF for 5 and 24 hr. Apoptosis was analyzed by annexin V staining.
(B) The cell lines in (A) were treated with 2 ng/ml of TNF for the indicated times. Cell extracts (150 μg) were analyzed by western blot as in Figure 1C. Anti-ZAP70 blotting confirmed equivalent loading.
(C) RIP1-null or RIP1-WT cells were transduced with control GST or TRAF2DN-encoding retroviruses. Stably selected cells were treated with indicated concentrations of TNF for 5 or 24 hr and stained with annexin V-PE.
(D) The data from (C) where the cells were stimulated with 5 ng/ml TNF for 24 hr are plotted in a bar graph to highlight the dual role of RIP1.
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5. Figure 4 Ubiquitination of RIP1 Regulates Association with CASPASE-8
(A) RIP1-WT and RIP1-K377R cells were stimulated with 10 ng/ml TNF for the indicated times. FLAG-tagged RIP1 and associated proteins were immunoprecipitated with anti-FLAG beads and eluted with the FLAG peptide. The immune complexes were blotted sequentially with anti-CASPASE-8 and anti-RIP1 as a loading control. The anti-CASPASE-8 blot was also overexposed so that the p43/p41 cleaved form could be visualized.
(B) A model for the role of RIP1 in TNF-mediated apoptosis. (I) In the early phase of TNFR1 signaling, TRAF2-mediated ubiquitination of lysine 377 functions as a brake to prevent RIP1 from associating with CASPASE-8. This does not require NF-κB-mediated gene transcription. (II) In the late phase of TNFR1 signaling, a second prosurvival signal provided by the ubiquitination of lysine 377 becomes effective via the upregulation of NF-κB-dependent antiapoptotic genes such as c-FLIP. (III) In the absence of K63-linked polyubiquitination, RIP1 becomes a death-signaling molecule by forming a complex with CASPASE-8 to initiate apoptosis. Red indicates antiapoptotic molecules, and green indicates proapoptotic molecules.
Current Biology  , DOI: ( /j.cub )
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