iκbα wt  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Analysis of Nuclear Factor-?B (NF-?B) Essential Modulator (NEMO) Binding to Linear and Lysine-linked Ubiquitin Chains and Its Role in the Activation of NF-?B *

doi: 10.1074/jbc.M112.347195

Figure Lengend Snippet: Linear di-ubiquitin is essential and sufficient to activate the IKK complex and NF-κB. A, linear di-ubiquitin and IKK complex induce phosphorylation of IκBα in vitro. Recombinant IKKα/β and NEMO (IKKγ) complex, MBP-IκBα-WT, or MBP-IκBα-AA, and linearly, Lys-63-, or Lys-48-linked di-ubiquitin were incubated as described under “Experimental Procedures.” Phosphorylation of MBP-IκBα by IKK was detected by immunoblotting. B, linear di-ubiquitin-fused NEMO induces sufficient NF-κB activity. HEK293T cells were transfected with increasing amounts (0.01, 0.03, 0.1, 0.3, and 1.0 μg) of FLAG-NEMO-[Ub]0–7 plasmids with NF-κB luciferase reporter, and the luciferase activity was measured 24 h after transfection. C, endogenous IKKα/β bound to linear di-ubiquitin-fused NEMO were phosphorylated. HEK293T cells, transfected with the indicated plasmids, were lysed and immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were separated by SDS gels and immunoblotted with anti-phospho-IKKα/β and anti-IKKα/β antibodies. D, linear ubiquitin-fused NEMO-F312A mutant does not induce sufficient NF-κB activity. HEK293T cells were transfected with increasing amounts (0.01, 0.03, 0.1, 0.3, and 1.0 μg) of FLAG-NEMO-[Ub]0–4 plasmids with NF-κB luciferase reporter, and the luciferase activity was measured 24 h after transfection.

Article Snippet: Full-length murine NEMO and human IκBα-WT (amino acids 1–54) and IκBα-AA (amino acid 1–54 S34A/S36A) were cloned into pMAL-C2x (New England Biolabs).

Techniques: In Vitro, Recombinant, Incubation, Western Blot, Activity Assay, Transfection, Luciferase, Immunoprecipitation, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Analysis of Nuclear Factor-?B (NF-?B) Essential Modulator (NEMO) Binding to Linear and Lysine-linked Ubiquitin Chains and Its Role in the Activation of NF-?B *

doi: 10.1074/jbc.M112.347195

Figure Lengend Snippet: The E3-ligase LUBAC activates the IKK complex and NF-κB in a NEMO-ubiquitin binding-dependent manner. A, ubiquitin ligase activity of LUBAC enhances IκBα phosphorylation. E1, UbcH5c, LUBAC, ubiquitin, and HeLa S-100 fraction II were incubated with MBP-IκBα-WT or MBP-IκBα-AA as described under “Experimental Procedures.” Phosphorylation of exogenous and endogenous IκBα was detected by immunoblotting. B, ubiquitin binding of NEMO is crucial for NF-κB activation by LUBAC. NEMO-deficient N-1 cells were transfected with plasmids with NF-κB luciferase reporter and with WT-NEMO or NEMO-F312A and either LUBAC or treated with TNFα as indicated, and the luciferase activity was measured 24 h after transfection.

Article Snippet: Full-length murine NEMO and human IκBα-WT (amino acids 1–54) and IκBα-AA (amino acid 1–54 S34A/S36A) were cloned into pMAL-C2x (New England Biolabs).

Techniques: Binding Assay, Activity Assay, Incubation, Western Blot, Activation Assay, Transfection, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: Analysis of Nuclear Factor-?B (NF-?B) Essential Modulator (NEMO) Binding to Linear and Lysine-linked Ubiquitin Chains and Its Role in the Activation of NF-?B *

doi: 10.1074/jbc.M112.347195

Figure Lengend Snippet: Selective Lys-63-linked ubiquitin chain-binding NEMO chimeras are impaired in NF-κB activation. A, NEMO-deficient MEFs, reconstituted with linear chain binding-deficient and Lys-63-binding-selective NEMO mutants are impaired in IκBα degradation upon TNFα stimulation. NEMO-deficient MEFs stably reconstituted with different NEMO mutants were untreated or treated with TNFα (20 ng/ml) for the indicated times. The lysates were separated by SDS-PAGE, and protein content was analyzed by Western blotting. B, NEMO-deficient MEFs reconstituted with linear chain binding-deficient and Lys-63 binding-selective NEMO mutants are hampered in nuclear translocation of p65 upon TNFα stimulation. NEMO-deficient MEFs stably reconstituted with WT-NEMO or different NEMO mutants were treated with TNFα (20 ng/ml) for the indicated times. Nuclear and the cytoplasmic fractions were separated by SDS-PAGE, and the presence of p65 in the nuclear fraction was analyzed by Western blotting. Densitometrical quantification of p65 in the nuclear fraction in the shown Western blot is indicated in the graphs, where the intensity for vector (Mock) containing cells was set to 1, and the intensity of the other cell lines was related to mock. The p65 signal was measured using the software ImageJ and normalized by PARP level. C, NEMO-deficient MEFs, reconstituted with linear ubiquitin chain binding-deficient but Lys-63 binding-selective NEMO mutants are impaired in the induction of NF-κB target gene expression. NEMO-deficient MEFs stably reconstituted with different NEMO mutants were treated with TNFα (20 ng/ml) for 30 minutes, and mRNA was extracted. Quantitative PCR was performed as described under “Experimental Procedures.” Ct values of target gene A20 were normalized to Ct values of β-actin. Induction of gene expression in a cell line was determined by comparison with the expression level of the untreated sample of the same cell line. Values shown are means ± S.E. (error bars) of at least three individual experiments. *, p < 0.05, significant difference between WT-NEMO-expressing and NEMO-deficient MEFs or NEMO mutants expressing MEFs.

Article Snippet: Full-length murine NEMO and human IκBα-WT (amino acids 1–54) and IκBα-AA (amino acid 1–54 S34A/S36A) were cloned into pMAL-C2x (New England Biolabs).

Techniques: Binding Assay, Activation Assay, Stable Transfection, SDS Page, Western Blot, Translocation Assay, Plasmid Preparation, Software, Expressing, Real-time Polymerase Chain Reaction

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Nuclear factor kappaB-activated monocytes contribute to pancreatic cancer progression through the production of Shh

doi: 10.1007/s00262-009-0783-7

Figure Lengend Snippet: Shh produced by LPS-stimulated THP-1 cells promotes proliferation of pancreatic cancer cells A, THP-1 cells were cultured in the presence or absence of LPS for 24 h. THP-1 cells were then washed intensively to eliminate LPS and the cell number was readjusted. AsPC-1 or SUIT-2 was cocultured with non-stimulated THP-1 cells, LPS-stimulated THP-1 cells or LPS-stimulated THP-1 cells with 5E1 or IgG antibody for 4 days. Total cell number was counted with a Coulter counter. The fractions of THP-1 cells and PDAC cells were determined using a FACSCaliburTM and the absolute number of PDAC cells was calculated. b AsPC-1 or SUIT-2 was transfected with siRNA for Shh or control siRNA by Lipofectamine for 36 h, and the expression of Shh mRNA was evaluated by real-time PCR (left panels). Shh-silenced AsPC-1 or SUIT-2 was cocultured with non-stimulated THP-1 cells, LPS-stimulated THP-1 cells, or LPS-stimulated THP-1 cells with 5E1 or IgG antibody for 4 days. Total cell number was counted with a Coulter counter. The fractions of THP-1 cells and PDAC cells were determined using a FACSCaliburTM and the absolute number of PDAC cells was calculated. Bars SD.*P < 0.05. c AsPC-1 or SUIT-2 was transfected with siRNA for Smo or control siRNA by Lipofectamine for 36 h, and the expression of Smo mRNA was evaluated by real-time PCR (left panels). Wild-type, control siRNA or Smo-siRNA transfected AsPC-1 or SUIT-2 was cocultured with LPS-stimulated THP-1 cells for 4 days. Total cell number was counted with a Coulter counter. The fractions of THP-1 cells and PDAC cells were determined using a FACSCaliburTM and the absolute number of PDAC cells was calculated. Bars SD. *P < 0.05

Article Snippet: The pCMV-IκBα wild type (WT) and pCMV-IκBα mutant were purchased from BD Biosciences/Clontech (Palo Alto, CA).

Techniques: Produced, Cell Culture, Transfection, Expressing, Real-time Polymerase Chain Reaction