2A). and IL-1 were from Peprotech (Rocky Hill, NJ), and 4-Hydroxytamoxifen (4OHT) was purchased from Sigma (St. Lois, MO). Plasmids Plasmids encoding K13 and 4-Hydroxytamoxifen (4OHT)-inducible K13-ERTAM, CYLD, EDAR (ectodermal dysplasia receptor) and NEMO have been described previously , , , , . Retroviral constructs expressing NEMO mutants defective in linear ubiquitination were kindly provided by Dr. Ivan Dikic (Goethe University Medical School). Recombinant retroviruses were generated and used Cefminox Sodium to generate polyclonal populations of stably transduced MEFs following selection with puromycin essentially as described previously . Luciferase Reporter Assay 293T cells were transfected in a 24-well plate with various test plasmids along with an NF-B luciferase reporter construct (75 ng/well) and a pRSV/LacZ (-galactosidase) reporter construct (75 ng/well) as described previously . Cells were lysed 24C36 h later, and extracts were used for the measurement of firefly luciferase and -galactosidase activities, respectively. Luciferase activity was normalized relative to the -galactosidase activity to control for the difference in the transfection efficiency. Transient transfection of MEFs and measurement of luciferase activity was performed essentially as described previously . Briefly, MEFs were transfected in duplicate using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in a 24-well plate with the various test plasmids along with an NF-B/luciferase reporter construct (75 ng/well) and a luciferase reporter construct (phRG-TK, 75 ng/well, Promega, Madison, WI). The cells were lysed 48 hours later, and extracts were used for the measurement of firefly and luciferase activities as described in the Dual-Luciferase? Reporter (DLR?) Assay system from Promega. Firefly luciferase activity was normalized relative to the luciferase activity to control for the difference in the transfection efficiency. Western Blot Western blot analysis was performed essentially as described previously . Primary antibodies used in these experiments were: NEMO, Total-IB, Rel B, TRAF6 (Santa Cruz Biotechnology, Santa Cruz, CA); tubulin, M2 FLAG (Sigma, St. Louis, MO), and phospho-TAK1, phospho-IB and A20 (Cell Signaling, Danvers, MA). A mouse monoclonal antibody against K13 (8F6) was raised in our laboratory. Cefminox Sodium NF-B DNA-binding Assays The NF-B subunit composition of the K13-induced NF-B complexes in wild-type and MEFs was decided with an NF-B ELISA kit (TransAM NF-B; Active Motif, Carlsbad, CA) according to the manufacturers instructions. The electrophoretic mobility shift assay was performed as described previously . Pathscan ELISA Assay The PathScan Phospho-IKK (Ser176/180), Phospho-IKK (Ser177/181) sandwich ELISA Kits and PathScan Phospho-IB (Ser32) sandwich ELISA Cefminox Sodium antibody pair (Cell Signaling, Danvers, MA) were used to detect endogenous levels of IKK, IKK and IB proteins when phosphorylated at Ser176/180, Ser177/181 and Ser32, respectively. Statistical Analyses Two-tailed paired Students test was used to test for differences between two groups. Differences with a p0.05 were considered as statistically significant. All experiments were repeated a minimum of three times with duplicate/triplicate samples. Results TRAF6 is not Required for K13-induced NF-B Activation Different members of the TRAF family are required for NF-B activation by distinct stimuli. Thus, while TRAF2 is known to be required for NF-B activation by TNF, TRAF6 has been implicated in the activation of this pathway signaling via interleukin 1 and Toll like receptors , . We have recently exhibited that TRAF2 is not involved in K13-induced NF-B activation . To rule out the involvement of TRAF6 in K13-induced NF-B activity, we transiently transfected and MEFs with an empty vector or Cefminox Sodium a K13 expression construct and examined the activation of a cotransfected NF-B-Luc reporter construct. As shown in Physique 1A, we observed near comparative K13-induced NF-B-Luc activity in the and MEFs. Essentially comparable results were obtained when the experiment was repeated using the K13-ERTAM construct followed by treatment with 4OHT (Fig. 1B). Finally, we generated stable populations of and MEFs expressing an empty vector or the K13-ERTAM construct. The mutated estrogen receptor (ERTAM) in the K13-ERTAM construct does not bind to its physiological ligand estrogen but binds with very high affinity to the synthetic ligand 4OHT (4-hydroxytamoxifen) and allows the control of K13 activity in a Cefminox Sodium 4OHT-dependent fashion . We treated the resulted cells with 4OHT to activate K13 activity and assessed the activation of the NF-B pathway by measuring the upregulation of A20, a protein known to be induced by K13-induced NF-B activity . As shown in Fig. 1C, treatment with 4OHT resulted in comparative upregulation of A20 in the and which argues against the involvement of TRAF6 in K13-induced NF-B activation. Open in a separate window Physique 1 TRAF6 is not required for K13-induced NF-B activation. A. and MEFs were transfected with a control JTK2 vector or vector encoding K13 along with an NF-B-Luc construct (75 ng/well) and a Renilla reporter construct (75.