Journal:

Article Title: The Fas-associated death domain protein suppresses activation of NF-?B by LPS and IL-1?

doi: 10.1172/JCI14774

Figure Lengend Snippet: Deletion of FADD enhances LPS- and IL-1β–induced NF-κB activity and NF-κB–dependent gene expression. FADD+/+ and FADD–/– MEF lysates were immunoblotted with anti-murine FADD antibody to confirm the genetic phenotype of these cells (a). Molecular mass (in kDa) is indicated. In other experiments, FADD+/+ and FADD–/– MEFs were treated for 4.5 hours with medium, LPS (100 ng/ml), or mIL-1β (10 ng/ml), lysed, and assayed for luciferase activity (b). Alternatively, MEFs were treated for 12 hours and the culture supernatants analyzed for IL-6 (c) or KC (d). Vertical bars represent mean (± SE) luciferase activity in arbitrary units (b) or pg/ml (c and d). *Significantly increased compared with FADD+/+ MEFs exposed to the same treatment.

Article Snippet: FADD +/+ and FADD –/– mouse embryo fibroblasts (MEFs) (generous gift of Wen-Chen Yeh, Amgen Institute, Toronto, Canada) were generated as previously described ( 15 ) and cultured in DMEM medium (BioWhittaker Inc.) enriched with 10% FBS, L -glutamine (2 mM), sodium pyruvate (1 mM), and nonessential amino acids, in the presence of penicillin (100 U/ml) and streptomycin (100 μg/ml).

Techniques: Activity Assay, Gene Expression, Luciferase

Journal:

Article Title: The Fas-associated death domain protein suppresses activation of NF-?B by LPS and IL-1?

doi: 10.1172/JCI14774

Figure Lengend Snippet: Reconstitution of FADD reverses the enhanced NF-κB activation and NF-κB–dependent gene expression in FADD–/– MEFs exposed to LPS or IL-1β. Lysates derived from FADD–/– MEFs stably transfected with either GFP vector alone or FADD were immunoblotted with anti-human FADD antibody to confirm expression (a). Molecular mass (in kDa) is indicated. In other experiments, FADD–/– MEFs expressing either GFP or FADD were treated for 4.5 hours with medium, LPS (100 ng/ml), or mIL-1β (10 ng/ml), lysed, and assayed for luciferase activity (b). Alternatively, MEFs were treated for 12 hours and the culture supernatants analyzed for IL-6 (c) or KC (d). Vertical bars represent mean (± SE) luciferase activity in arbitrary units (b) or pg/ml (c and d). *Significantly decreased compared with GFP-expressing cells exposed to the same treatment.

Article Snippet: FADD +/+ and FADD –/– mouse embryo fibroblasts (MEFs) (generous gift of Wen-Chen Yeh, Amgen Institute, Toronto, Canada) were generated as previously described ( 15 ) and cultured in DMEM medium (BioWhittaker Inc.) enriched with 10% FBS, L -glutamine (2 mM), sodium pyruvate (1 mM), and nonessential amino acids, in the presence of penicillin (100 U/ml) and streptomycin (100 μg/ml).

Techniques: Activation Assay, Gene Expression, Derivative Assay, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay

Journal:

Article Title: The Fas-associated death domain protein suppresses activation of NF-?B by LPS and IL-1?

doi: 10.1172/JCI14774

Figure Lengend Snippet: Deletion of FADD enhances LPS- and IL-1β–induced degradation of IκB. FADD+/+ and FADD–/– MEFs were incubated with medium, LPS (100 ng/ml), or IL-1β (10 ng/ml) for increasing exposure times, and lysates derived from these cells were immunoblotted with antibodies raised against either IκB-α or IκB-β (a and b). In other experiments, FADD+/+ MEFs stably expressing GFP (+/+ GFP) or FADD–/– MEFs stably expressing either GFP (–/– GFP) or FADD (–/– + FADD) were treated with LPS (100 ng/ml) or IL-1β (10 ng/ml) for 45 minutes, and lysates were immunoblotted as above (c).

Article Snippet: FADD +/+ and FADD –/– mouse embryo fibroblasts (MEFs) (generous gift of Wen-Chen Yeh, Amgen Institute, Toronto, Canada) were generated as previously described ( 15 ) and cultured in DMEM medium (BioWhittaker Inc.) enriched with 10% FBS, L -glutamine (2 mM), sodium pyruvate (1 mM), and nonessential amino acids, in the presence of penicillin (100 U/ml) and streptomycin (100 μg/ml).

Techniques: Incubation, Derivative Assay, Stable Transfection, Expressing