Journal:

Article Title: FADD Negatively Regulates Lipopolysaccharide Signaling by Impairing Interleukin-1 Receptor-Associated Kinase 1-MyD88 Interaction ▿

doi: 10.1128/MCB.00600-07

Figure Lengend Snippet: FADD acts upstream of TRAF6 and associates with MyD88 and IRAK1. (A) Endothelial cells transduced with the empty vector (HMEC-MIY) or FADD (HMEC-FADD) were either left untreated or stimulated with LPS (100 ng/ml) for the indicated times (in minutes). Whole-cell lysates were prepared and immunoprecipitated with anti-TRAF6 antibody under denaturing conditions followed by immunoblotting with antiubiquitin and anti-TRAF6 antibodies. Lanes A are a negative control which contained the immunoprecipitating antibody with lysis buffer. Lanes L are a positive control and contain 30 μg of unstimulated protein lysate. Molecular mass (in kilodaltons) is shown to the left of the blots. IP, immunoprecipitation; WB, Western blotting or immunoblotting; (Ub)n TRAF6, polyubiquitinated TRAF6. Data are representative of three independent experiments. (B) (Top) Coimmunoprecipitation analysis of lysates of 293T cells cotransfected with the indicated combinations of epitope-tagged expression plasmids (Myc-tagged MyD88 [Myc-MyD88] and Flag-tagged FADD [Flag-FADD]) and immunoprecipitated (IP) with an anti-Myc antibody. (Bottom) Immunoblot (IB) of whole-cell lysates, using anti-Flag or anti-Myc antibodies. NS, nonspecific band recognized by anti-Flag antibody. (C) (Top) Coimmunoprecipitation analysis of lysates of 293T cells cotransfected with the indicated combinations of epitope-tagged expression plasmids (Flag-tagged IRAK1 [Flag-IRAK1] and Flag-FADD) and immunoprecipitated with an anti-IRAK1 antibody. (Bottom) Immunoblot of whole-cell lysates using anti-Flag antibody. NS, nonspecific band recognized by anti-Flag antibody. (D) (Top) Coimmunoprecipitation analysis of lysates of 293T cells cotransfected with the indicated combinations of epitope-tagged expression plasmids (Flag-IRAK1 and AU1-tagged FADD [AU1-FADD]) and immunoprecipitated with an anti-FADD antibody or control IgG. (Bottom) Whole-cell lysates were immunoblotted with anti-FADD or anti-IRAK1 antibodies. (E) (Top) Coimmunoprecipitation analysis of lysates of 293T cells transfected with empty vector (pcDNA3) or the indicated epitope-tagged expression plasmid (AU1-FADD, Flag-FADD-DED, AU1-FADD-DD, or Flag-IRAK1) and immunoprecipitated with an anti-IRAK1 antibody or control IgG. (Bottom) Whole-cell lysates were immunoblotted with anti-FADD or anti-Flag antibodies. Molecular mass (in kilodaltons) is shown to the left of the blots.

Article Snippet: Anti-MyD88 antibody was purchased from Alexis Biochemicals (Lausanne, Switzerland).

Techniques: Transduction, Plasmid Preparation, Immunoprecipitation, Western Blot, Negative Control, Lysis, Positive Control, Expressing, Transfection

Journal:

Article Title: FADD Negatively Regulates Lipopolysaccharide Signaling by Impairing Interleukin-1 Receptor-Associated Kinase 1-MyD88 Interaction ▿

doi: 10.1128/MCB.00600-07

Figure Lengend Snippet: FADD interferes with IRAK1-MyD88 interaction. (A) Endothelial cells expressing the empty vector (HMEC-MIY) or FADD (HMEC-FADD) were either unstimulated or stimulated with LPS (100 ng/ml) for the indicated times (in minutes). Cell lysates were immunoprecipitated (IP) with anti-IRAK1 antibody and then immunoblotted with antibodies against MyD88, TRAF6, FADD, and IRAK1. (B) Lysates from endothelial cells were prepared as described above for panel A and subjected to immunoprecipitation (IP) with anti-FADD antibody, followed by immunoblotting for IRAK1, MyD88, and FADD. (C) MEF expressing wild-type FADD (MEF-FADD-WT) or MEF deficient in FADD (MEF-FADD-KO) were stimulated with LPS (1 μg/ml) for the indicated times (in minutes). Cell lysates were subjected to anti-IRAK1 immunoprecipitation, followed by immunoblotting for MyD88, TRAF6, ubiquitin, FADD, and IRAK1. Molecular mass (in kilodaltons) is shown to the left of the blots. Ubn-TRAF6, polyubiquitinated TRAF6.

Article Snippet: Anti-MyD88 antibody was purchased from Alexis Biochemicals (Lausanne, Switzerland).

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal:

Article Title: FADD Negatively Regulates Lipopolysaccharide Signaling by Impairing Interleukin-1 Receptor-Associated Kinase 1-MyD88 Interaction ▿

doi: 10.1128/MCB.00600-07

Figure Lengend Snippet: IRAK1 is required for FADD-MyD88 interaction. (A) MEF expressing wild-type MyD88 (MEF-MydD88-WT) or MEF lacking MyD88 (MEF-MydD88-KO) were stimulated with LPS (1 μg/ml) as indicated, and cell lysates were immunoprecipitated with anti-FADD antibody. Coimmunoprecipitating IRAK1 and MyD88 were detected by immunoblotting with anti-IRAK1 and anti-MyD88 antibodies, respectively. (B) Lysates from IRAK1-deficient MEF (MEF-IRAK-1-KO) or MEF expressing wild-type IRAK1 (MEF-IRAK-1-WT) controls were prepared and subjected to immunoprecipitation with anti-FADD antibody. Coimmunoprecipitating MyD88 and IRAK1 were detected using anti-MyD88 and anti-IRAK1 antibodies, respectively. IP, immunoprecipitation, NS, nonspecific band recognized by antibody.

Article Snippet: Anti-MyD88 antibody was purchased from Alexis Biochemicals (Lausanne, Switzerland).

Techniques: Expressing, Immunoprecipitation, Western Blot

Journal:

Article Title: FADD Negatively Regulates Lipopolysaccharide Signaling by Impairing Interleukin-1 Receptor-Associated Kinase 1-MyD88 Interaction ▿

doi: 10.1128/MCB.00600-07

Figure Lengend Snippet: FADD attenuates signaling only through IRAK1/MyD88-dependent receptors. MEF expressing wild-type FADD (FADD-WT) or deficient in FADD (FADD-KO) were stimulated with Pam3CSK4 (500 ng/ml) (A), IL-1β (50 ng/ml) (B), and poly(I)·poly(C) (Poly I:C) (100 μg/ml) (C) for the indicated times (in minutes), and lysates were immunoblotted for phospho- and total JNK and Akt. Results are representative of three independent experiments. pJNK, phospho-JNK; pAkt, phospho-Akt.

Article Snippet: Anti-MyD88 antibody was purchased from Alexis Biochemicals (Lausanne, Switzerland).

Techniques: Expressing