Figure 2
Figure 2. RIP1 is required for the antiapoptotic function of Toso and Toso facilitates RIP1 ubiquitination in response to CD95 stimulation. (A) Wild-type or RIP1 deficient Jurkat cells were transfected with a control or a Toso construct. Cells were stimulated with CD95L for 5 hours and apoptosis was assessed by annexin V staining. RIP1 (top) and actin (bottom) expression levels in cell lysates are shown by Western blotting. Data are representative of 3 experiments. Statistical analysis: Student t test (*P < .05, **P < .01). (B) BJAB cells stably expressing Toso were transfected with 3 different RIP1 specific shRNA constructs or mock treated. CD95-induced apoptosis was analyzed as in panel A. The knock-down efficiency of RIP1 was monitored by immunoblotting for RIP1 (top). Actin was used as a control for protein loading (bottom). Data are representative of 2 experiments. (C) Jurkat cells were transiently transfected with a control or a Toso construct. Cells were stimulated with CD95L for the indicated times and ubiquitinated proteins were isolated by immunoprecipitation (IP) using an antiubiquitin antibody. Ubiquitinated endogenous RIP1 was detected in immunoprescipitates using RIP1 antibody (top) and total lysates were probed for actin (bottom). Data are representative of 2 experiments. (D) The antiapoptotic function of Toso depends on RIP1 ubiquitination at lysine 377. Wild-type Jurkat cells were transiently transfected with control pIRES-GFP vector (lane 1), Toso-IRES-GFP only (lane 2), wt RIP1-FLAG and Toso-IRES-GFP (lane 3), or RIP1-K377R-FLAG (K/R) and Toso-IRES-GFP (lane 4). Cells were stimulated with CD95L for 5 hours. Apoptotic cells in the GFP+ population were determined by annexin V staining. Representative data from 5 independent experiments are shown. Statistical analysis: Student t test (*P < .05, **P < .01). (E) RIP1 deficient Jurkat (RIP1−/−) cells were transiently transfected with control pIRES-GFP vector (lane 1), wt RIP1-FLAG and control pIRES-GFP vector (lane 2), Toso-IRES-GFP only (lane 3), wt RIP1-FLAG and Toso-IRES-GFP (lane 4), RIP1-K377R-FLAG (K/R) and control pIRES-GFP vector (lane 5), or RIP1-K377R-FLAG (K/R) and Toso-IRES-GFP (lane 6). Cells were stimulated with CD95L for 5 hours. Apoptotic cells in the GFP+ population were determined by annexin V staining. Representative data from 3 independent experiments are shown.

RIP1 is required for the antiapoptotic function of Toso and Toso facilitates RIP1 ubiquitination in response to CD95 stimulation. (A) Wild-type or RIP1 deficient Jurkat cells were transfected with a control or a Toso construct. Cells were stimulated with CD95L for 5 hours and apoptosis was assessed by annexin V staining. RIP1 (top) and actin (bottom) expression levels in cell lysates are shown by Western blotting. Data are representative of 3 experiments. Statistical analysis: Student t test (*P < .05, **P < .01). (B) BJAB cells stably expressing Toso were transfected with 3 different RIP1 specific shRNA constructs or mock treated. CD95-induced apoptosis was analyzed as in panel A. The knock-down efficiency of RIP1 was monitored by immunoblotting for RIP1 (top). Actin was used as a control for protein loading (bottom). Data are representative of 2 experiments. (C) Jurkat cells were transiently transfected with a control or a Toso construct. Cells were stimulated with CD95L for the indicated times and ubiquitinated proteins were isolated by immunoprecipitation (IP) using an antiubiquitin antibody. Ubiquitinated endogenous RIP1 was detected in immunoprescipitates using RIP1 antibody (top) and total lysates were probed for actin (bottom). Data are representative of 2 experiments. (D) The antiapoptotic function of Toso depends on RIP1 ubiquitination at lysine 377. Wild-type Jurkat cells were transiently transfected with control pIRES-GFP vector (lane 1), Toso-IRES-GFP only (lane 2), wt RIP1-FLAG and Toso-IRES-GFP (lane 3), or RIP1-K377R-FLAG (K/R) and Toso-IRES-GFP (lane 4). Cells were stimulated with CD95L for 5 hours. Apoptotic cells in the GFP+ population were determined by annexin V staining. Representative data from 5 independent experiments are shown. Statistical analysis: Student t test (*P < .05, **P < .01). (E) RIP1 deficient Jurkat (RIP1−/−) cells were transiently transfected with control pIRES-GFP vector (lane 1), wt RIP1-FLAG and control pIRES-GFP vector (lane 2), Toso-IRES-GFP only (lane 3), wt RIP1-FLAG and Toso-IRES-GFP (lane 4), RIP1-K377R-FLAG (K/R) and control pIRES-GFP vector (lane 5), or RIP1-K377R-FLAG (K/R) and Toso-IRES-GFP (lane 6). Cells were stimulated with CD95L for 5 hours. Apoptotic cells in the GFP+ population were determined by annexin V staining. Representative data from 3 independent experiments are shown.

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