Suppression of TNF-α-Induced Apoptosis by NF-κB

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IκBαM was generated by digestion of plasmids pCMX-IκBαS32/36A and pCMX-IκBαMutF with Eco NI and Bst EII and ligation of the small S32/36A fragment to the large vector/IκBαMutF fragment. The resulting expression plasmid, called pCMX-IκBαM, was confirmed by in vitro transcription and translation (TNT, Promega) with the T7 promoter present in pCMX followed by immunoprecipitation with IκBα-specific antibody. pCMX-IκBαS32/36A was constructed by removal of the Bam HI-Hind III fragment of pBS-IκBαS32/36A and ligation of the fragment into the Bam HI-Hind III sites in pCMX. pBS-IκBαS32/36A was constructed by site-directed mutagenesis of the plasmid pBS-IκBαand confirmed by sequencing. The construction of pCMX-IκBαMutF has been described (5). pLIκBαMSN was constructed by blunt insertion of the Eco RV fragment from pCMX-IκBαM into the Hpa I site of pLXSN (25)

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Mouse embryo fibroblasts were derived from Swiss Webster mice as described (26) and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Human embryo fibroblasts were prepared and grown identically to MEF cells. Two hundred ninety-three cells expressing gag and pol were grown in DMEM, 10% FBS, and selection was maintained with blastacidin (20 µg/ml). T24 cells (American Type Culture Collection) and Jurkat cells were grown in RPMI 1640 in 10% FBS. All the pools of stable cells were produced in a similar manner. First, 293 cells stably expressing Moloney gag and pol under control of cytomegalovirus (CMV) promoter-enhancer were transiently transfected by CaPO₄ precipitation with 20 µg of pLXSN retroviral vector (3) and 5 µg of the plasmid pMDG (27) containing the vesicular stomatitis virus (VSVg) envelope downstream of the CMV promoter-enhancer. After 48 and 72 hours, the medium was removed, filtered, and either stored at −20°C or used immediately for infection. Infection was performed on ~0.5 × 10⁵ cells in 3 ml with Polybrene (8 µg/ml) for 8 to 12 hours. Cells were allowed to expand for 48 hours and were then selected for neomycin resistance. Amounts of G418 used in selecting the various cell types were as follows: MEF and HEF cells, 800 µg/ml; T24, 400 µg/ml; Jurkat, 1 mg/ml. Immunoblotting was then performed to analyze the expression of IκBαM

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For immunoblot analysis, cytoplasmic extracts were prepared as described (28), and 50 µg (as determined by Bradford analysis) was applied to 12% SDS-PAGE gels and transferred to 0.2-µm pore nitrocellulose membranes (Schleicher & Schuell). Blots were probed for 12 hours with antibody raised against the NH₂-terminus of IκBα (C-15, Santa Cruz Biotech) and diluted 1:1000 in phosphate-buffered saline (PBS) with 0.2% Tween-20 (Sigma) and 5% nonfat milk (Carnation). After washing, the blots were probed with horseradish peroxidase (HRP)-conjugated donkey antiserum to rabbit immunoglobulin G (Amersham) diluted 1:3000 for 2 hours. Bands were visualized by use of the Renaissance chemiluminescence kit (Dupont)

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Electrophoretic mobility-shift assay was done as described (29). Briefly, 5 µg of nuclear extract were mixed with 0.5 µg of poly(dI-dC) and DNA binding buffer in a total volume of 10 µl and incubated on ice for 20 min. ³²P-labeled oligonucleotides (6000 cpm per sample) were then added to the binding reaction and incubated for 30 min at room temperature. Competition was performed by addition of 10 ng of unlabeled oligonucleotide (500-fold excess) during the incubation on ice followed by addition of labeled oligonucleotide. The HIV-κB oligonucleotide has been described (30). Oct-1 and AP-1 consensus oligonucleotides were purchased from Santa Cruz Biotech. All the oligonucleotides were end-labeled by use of T4 polynucleotide kinase and [γ-³²P]adenosine triphosphate (Dupont NEN). TNF-α, IL-1α, phorbol 12-myristate 13-acetate (PMA), and ionomycin A23187 were obtained from Calbiochem

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Quantitation of apoptosis by annexin V binding was done as described (12). Briefly, MEF cells were plated onto 24-well plates and treated under the conditions indicated. The cells were trypsinized and resuspended in the original supernatant to ensure that both attached and nonattached cells were analyzed. The cells were washed once in PBS and resuspended in annexin V binding buffer. Fluorescein isothiocyanate (FITC)-conjugated annexin V was added, and the samples were analyzed by flow cytometry with a FACScan (Becton-Dickinson). Jurkat cells were plated onto 24-well dishes at an initial density of 105 per milliliter and treated identically to MEF cells. Quantitation was done with Cell Quest software

Van Antwerp D. J., Martin S. J., Green D. R., Verma I. M., unpublished results.

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We are grateful to B. Sha, A. Beg, and D. Baltimore for p50 and RelA knockout fibroblast cell lines and to D. Trono for Jurkat cells. We thank members of the Verma laboratory for helpful discussions, P. Charon for help in manuscript preparation, and D. Finucane for assistance with flow cytometry. D.J.V. is a graduate student in the Department of Chemistry and Biochemistry at the University of California, San Diego, and is supported by a fellowship from the Chapman Charitable Trust. S.J.M. is a Wellcome Trust Senior Fellow. T.K. is supported by a European Molecular Biology Organization Fellowship. D.R.G.'s laboratory is supported by grants GM52735 from the National Institutes of Health and CB-82 from the American Cancer Society. I.M.V.'s laboratory is supported by grants from the NIH and American Cancer Society. I.M.V. is an American Cancer Society Professor of Molecular Biology