of recombinant mouse TNF 2.5 ng/ml (BD Biosciences). Mouse B cell line A20 and B cell hybridoma SP2/0 were cultured in RPMI containing 10% heat-inactivated foetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 50 mM 2-mercaptoetha- nol, in the presence or absence of mTNF 20 ng/ml for 8 days at 37uC and 5% CO2. Cell Stimulation and IL-2 assay 11A2 cells were harvested, washed and resuspended in complete medium at 106 cells/ml in the absence of TNF, prior to incubation with either plate-bound anti-CD3e, or with PMA and ionomycin (Calbiochem, Merck Biosciences, Beeston, UK) at the concentrations and for the times indicated. IL-2 protein in cell supernatants was assayed by fluorescent immunosorbant assay, as described [41]. Ribonuclease protection assay and RNA stability Control and TNF-treated 11A2 cells were resuspended in complete medium at 106 cells/ml in the absence of TNF, then incubated with either plate-bound anti-CD3e 10 mg/ml, or PMA and ionomycin for the times indicated. Cells were harvested, washed in ice-cold PBS and total RNA extracted (QIAampH RNA Blood Mini Kit, Qiagen, Crawley, UK). mRNA species were visualised by ribonuclease protection assay (BD RiboQuantTM kit mCK-1, BD Biosciences) and phosphor-imaging (Fuji FLA 2000). For stability studies, actinomycin D 10 mg/ml was added after 4 hours’ stimulation, and cells were harvested at the times indicated, washed in ice-cold PBS and snap-frozen until RNA was extracted and analysed. Transfections and reporter assays Control and TNF-treated 11A2 T cells (561062107 cells per point) were transfected using AmaxaH NucleofectorH I, Kit VH, programme A10 (AmaxaH, Lonza Wokingham, Ltd, UK) with 10 mg firefly luciferase reporter vectors pAP-1-TA-luc (TRE consensus sequence), pNFAT-TA-luc (ARRE-2 from mouse pIL- 2), or pNF-kB-TA-luc (consensus response element of mouse MHCII invariant chain) (all from Takara Bio Europe/Clontech, Lonza Wokingham, Ltd, UK). In all cases, cells were co- transfected with 0.2 mg phRL-SV40 expressing Renilla luciferase (Promega UK), to normalise data for transfection efficiency. Note: Among many other transcription factor binding sites, the SV40 promoter/enhancer of phRL-SV40 contains two NF-kB consen- sus sequences with the potential for interference in NF-kB- dependent promoter assays. However, transfecting with the lowest feasible amount of phRL-SV40 DNA, we found the stimulation index (due to PMA and ionomycin) of renilla luciferase expression was similar for control and TNF-treated cells in all experiments. Thus any differences seen between control and TNF-treated cells in terms of firefly luciferase normalised to renilla luciferase expression were independent of effects of TNF pre-treatment on phRL-SV40. Transfected cells were plated out at 106/ml, allowed to recover for two hours, then stimulated with PMA 50 ng/ml and ionomycin 100 ng/ml for up to 6 hours. Cell lysates were assayed for luminescence due to firefly and renilla luciferase activities (Dual-Luciferase ReporterTM assay, Promega UK). Generation of pFLAG-CMV4-c-Rel The murine c-Rel sequence was digested from cloning vector pSPORT (Invitrogen, Paisley, UK), end-filled to allow cloning into expression vector pFLAG-CMV4 (Sigma-Aldrich) and repaired for a ‘stop’ codon mutation at the end of the rel homology domain before sequencing across pFLAG-c-Rel (Eurofins MWG Operon, Regulation of c-Rel by IkBe in Murine Lymphocytes London, UK). Expression of c-Rel and HA immunoreactivity of the correct size was confirmed by immunoblot following lipofection of NIH-3T3 fibroblasts. For reconstitution experiments in 11A2 cells, 2 mg of pFLAG-CMV4-c-Rel or empty vector were used. EMSAs, immunoblots, and immunoprecipitations Control and TNF-treated cells (106/ml) were stimulated with PMA 10 and ionomycin 50 ng/ml, or PMA 50 and ionomycin 100 ng/ml for the times indicated. Cells were harvested and washed in ice-cold PBS. Cytoplasmic and nuclear extracts were prepared by a method adapted from Dean et al [44]: 56106 cells were resuspended in 500 ml hypotonic lysis buffer (10 mM HEPES pH 7.6, 40 mM KCl, 3 mM MgCl2, 5% glycerol, 1% Triton X- 100, 2 mM DTT, 2 mM NaF, 1 mM Na3VO4, 25 mM b- glycerophosphate and protease inhibitor cocktail 10 ml/ml) for 5 minutes on ice. After centrifugation at 500 g x 3 minutes at 4 uC, the supernatant was saved (cytosolic fraction), and pelleted nuclei washed in 250 ml wash buffer (10 mM HEPES pH 7.6, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 2 mM NaF, 1 mM Na3VO4, 25 mM b-glycerophosphate and protease inhibitor cocktail 10 ml/ ml). A sample was examined for successful nuclear extraction of intact nuclei by light microscopy. Nuclei were pelleted as before then extracted in 150 ml nuclear extraction buffer (20 mM HEPES pH 7.6, 420 mM NaCl, 1.5 mM MgCl2, 25% glycerol, 1 mM DTT, 0.2 mM EDTA, 2 mM NaF, 1 mM Na3VO4, 25 mM b- glycerophosphate and protease inhibitor cocktail 10 ml/ml) for 15 minutes on ice, subjected to one freeze-thaw cycle at 280 uC, and clarified by centrifugation at 14,0006g for 15 minutes at 4 uC. Nuclear extract and cytosol were stored at 280 uC until use. For immunoblot, up to 20 mg nuclear or cytosolic proteins were separated by SDS-PAGE (NuPAGE Bis-Tris MOPS system, Invitrogen) and proteins of interest detected by immunoblot and ECL (GE Healthcare Life Sciences, Amersham, UK). Immunoblots of all nuclear and cytoplasmic proteins were probed for b-actin, which is abundant in both nucleus and cytoplasm [45,46], and a- tubulin, abundant in cytoplasm only. Actin detected in the absence of tubulin indicated freedom of nuclear extracts from cytosolic contamination and was used to normalise for protein loading in both nuclear and cytosolic immunoblots. For EMSA, 5 mg nuclear proteins were incubated with 32P end-labelled oligonucleotide probe for 15 minutes at room temperature then separated in a 6% non- denaturing polyacrylamide gel, the gel dried, and protein-bound oligonucleotide detected by phosphor-imaging and by exposure of photographic film to dried gel. For probe competition, cold probe was added to the incubation in 100-fold excess. For super-shifting assays, proteins were incubated with 1 mg antibody for 10 minutes at room temperature prior to addition of radiolabelled probe. For immunoprecipitations, 56106 cells were lysed in 500 ml 50 mM TRIS.HCl pH 8, 200 mM NaCl, 1% TX-100, 1 mM DTT, 0.1 mM EDTA, 1 mM Na3VO4, 50 mM NaF, 10 mM b- glycerophosphate, and protease inhibitor cocktail 10 ml/ml. IkB and NF-kB proteins were immunoprecipitated from 100 mg clarified lysate and analysed by immunoblot for the target and co- immunoprecipitated proteins. IkBe2/2 mice The IkBe coding sequence was replaced by an nlslacZ/IRESneo construct and fully back-crossed onto the C57BL/6J background as reported previously by Me´met and colleagues [47]. Mice were bred and housed in a specific pathogen-free environment and genotyped by PCR, as described (30). IkBe +/+ wild-type, +/2 heterozygote, or 2/2 homozygote knockout (.7 weeks or ,18 weeks old) male mice were used in all experiments unless otherwise stated. Where WT mice were not littermates of IkBe2/2 PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e24504