{"id":573,"date":"2011-04-26T15:20:55","date_gmt":"2011-04-26T19:20:55","guid":{"rendered":"https:\/\/www.bu.edu\/nf-kb\/the-gilmore-lab\/data-link-12\/"},"modified":"2011-04-26T15:20:55","modified_gmt":"2011-04-26T19:20:55","slug":"data-link-12","status":"publish","type":"page","link":"https:\/\/www.bu.edu\/nf-kb\/the-gilmore-lab\/data-link-12\/","title":{"rendered":"Data Link 12"},"content":{"rendered":"

Appendix of Plasmids and Primers for <\/strong><\/p>\n

Garbati MR and TD Gilmore (2008) Ser484 and Ser494 in REL are the major sites of IKK phosphorylation in vitro: evidence that IKK does not directly enhance GAL4-REL transactivation. Gene Expression 14: 195-205<\/p>\n

pGEM-based Plasmids <\/span><\/strong><\/p>\n

pGEM4:<\/span> Cloning vector for in vitro transcription\/translation with either SP6 or T7 promoter elements (Promega)<\/p>\n

pGEM-Hu-cRel:<\/span> pGEM4 containing wild-type REL; an XbaI-XhoI\/Klenow fragment of REL was subcloned into pGEM4 digested with XbaI-HincII (Barkett et al, 2001)<\/p>\n

pGEM-REL-S484,494A:<\/span> pGEM4 containing REL-S484,494A; an EcoRV-NdeI fragment from JD-REL-S484,494A was subcloned into pGEM-Hu-cRel digested with EcoRV-NdeI <\/span><\/strong><\/p>\n

Retroviral Vectors (used for subcloning) <\/span><\/strong><\/p>\n

JD214 BS+:<\/span> Spleen necrosis virus vector (Sif et al, 1993)<\/p>\n

JD-REL:<\/span> JD214 BS+ containing wild-type REL; an XbaI-XhoI REL fragment was subcloned into JD214 BS+ digested with XbaI-SalI (Gilmore et al, 2001)<\/p>\n

JD-REL-S484A:<\/span> JD214 BS+ containing REL-S484A; an ApaI-HindIII fragment from pGEX-3\u2019REL-S484A was subcloned into JD-REL digested with ApaI-HindIII<\/p>\n

JD-REL-S484,494A:<\/span> JD214 BS+ containing REL-S484,494A; an ApaI-HindIII fragment from pGEX-3\u2019REL-S484,494A was subcloned into JD-REL digested with ApaI-HindIII<\/p>\n

JD-REL-S484,494D:<\/span> JD214 BS+ containing REL-S484,494D; an EcoRV-NdeI fragment from pSG-REL-S484,494D was subcloned into JD-REL digested with EcoRV-NdeI<\/p>\n

pcDNA-based Expression Vectors<\/span> <\/strong><\/p>\n

pcDNA 3.1(-):<\/span> CMV promoter-driven expression vector (Invitrogen)<\/p>\n

pcDNA-REL:<\/span> pcDNA containing wild-type REL; an XbaI-HindIII REL fragment was subcloned into pcDNA 3.1(-) digested with XbaI-HindIII (Leeman et al, 2008)<\/p>\n

pcDNA3-FLAG:<\/span> pcDNA containing a FLAG tag upstream of the multiple cloning site (MCS)<\/p>\n

pcDNA-FLAG-RHD-RID:<\/span> pcDNA containing FLAG-tagged REL aa 1-423; a PCR fragment from pGEM-Hu-cRel containing REL aa 1-423 was digested with EcoRI-BamHI and subcloned into pcDNA3-FLAG digested with EcoRI-BamHI (created by M. Garbati for Leeman et al, 2008)<\/p>\n

pcDNA-FLAG-REL:<\/span> pcDNA containing FLAG-tagged REL; an EcoRV-XhoI fragment from pGEM-Hu-cRel was subcloned into pcDNA-FLAG-RHD-RID digested with EcoRV-XhoI (created by M. Garbati for Leeman et al, 2008)<\/p>\n

pcDNA-FLAG-REL-S484,494A:<\/span> pcDNA containing FLAG-tagged REL-S484,494A; an EcoRV-XhoI fragment from pGEM-REL-S484,494A was subcloned into pcDNA-FLAG-RHD-RID digested with EcoRV-XhoI<\/p>\n

pcDNA-REL-S484,494D:<\/span> pcDNA containing REL-S484,494D; an EcoRV-HindIII fragment from JD-REL-S484,494D was subcloned into pcDNA-REL digested with EcoRV-HindIII<\/p>\n

pcDNA-FLAG-IKKa:<\/span> pcDNA containing FLAG-tagged human IKKa (Starczynowski et al, 2007)<\/p>\n

pcDNA-FLAG-IKKb:<\/span> pcDNA containing FLAG-tagged human IKKb (Liang et al, 2003)<\/p>\n

pcDNA-FLAG-IKKb-S177,181E:<\/span> pcDNA containing FLAG-tagged human IKKb-S177,181E (Liang et al, 2006)<\/p>\n

pcDNA-HA-IKKb-S177,181E:<\/span> pcDNA containing HA-tagged human IKKb-S177,181E (gift of Sankar Ghosh, Yale University)<\/p>\n

pcDNA-FLAG-IkBa-S32,36A:<\/span> pcDNA containing FLAG-tagged human IkBa-S32,36A (gift of Susan Kandarian, Boston University)<\/p>\n

pcDNA-mouse-RelA:<\/span> pcDNA containing mouse RelA (Liang et al, 2006)<\/p>\n

<\/span><\/strong><\/p>\n

pGEX-based GST Fusion Protein Bacteria Expression Vectors<\/span><\/strong><\/p>\n

pGEX KG:<\/span> Expression plasmid containing GST domain upstream of MCS<\/p>\n

pGEX-3\u2019REL:<\/span> C-terminal sequences of wild-type REL (aa 324-587) fused to GST; a PCR fragment using pGEM-Hu-cRel as a template was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII (created by M. Garbati for Straczynowski et al, 2007)<\/p>\n

pGEX-3\u2019REL-S484A:<\/span> REL-S484A (aa 323-587) fused to GST; overlapping PCR mutagenesis using pSG-REL-S484P as a template was used to mutate REL codon 484 to an Ala codon.\u00a0 The fragment containing REL-S484A was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII<\/p>\n

pGEX-3\u2019REL-S494A: <\/span> REL-S494A (aa 323-587) fused to GST; overlapping PCR mutagenesis using pGEM-Hu-cRel as a template was used to mutate REL codon 494 to an Ala codon.\u00a0 The fragment containing REL-S494A was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII<\/p>\n

pGEX-3\u2019REL-S484,494A:<\/span> REL-S484,494A (aa 323-587) fused to GST; overlapping PCR mutagenesis using pGEX-3\u2019REL-S484A as a template was used to mutate REL codon 494 to an Ala codon.\u00a0 The fragment containing REL-S484,494A was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII<\/p>\n

pGEX-3\u2019RELD58:<\/span> REL aa 323-529 fused to GST; a PCR fragment from pGEM-RELD58 (Starczynowski et al, 2003) was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII<\/p>\n

pGEX-3\u2019RELD90:<\/span> REL aa 323-497 fused to GST; a PCR fragment from pGEM-RELD90 (Starczynowski et al, 2003) was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII<\/p>\n

pGEX-3\u2019RELD110:<\/span> REL aa 323-477 fused to GST; a PCR fragment from pGEM-RELD110 (Starczynowski et al, 2003) was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII<\/p>\n

pGEX-3\u2019RELD132:<\/span> REL aa 323-455 fused to GST; a PCR fragment from pGEM-RELD132 (Starczynowski et al, 2003) was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII<\/p>\n

pGEX-REL-aa-476-504:<\/span> REL aa 476-504 fused to GST; a PCR fragment containing REL aa 476-504 was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII<\/p>\n

pGEX-REL-aa-476-504-S484A<\/span>:\u00a0 REL aa 476-504 S484A fused to GST; a PCR fragment containing REL aa 476-504 S484A was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII<\/p>\n

pGEX-REL-aa-476-504-S494A<\/span>:\u00a0 REL aa 476-504 S494A fused to GST; a PCR fragment containing REL aa 476-504 S494A was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII<\/p>\n

pGEX-REL-aa-476-504-S484,494A:<\/span> REL aa 476-504 S484,494A fused to GST; a PCR fragment containing REL aa 476-504 S484,494A was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII <\/span><\/strong><\/p>\n

Expression Vectors for GAL4 Fusion Proteins in A293 Cells <\/span> <\/strong><\/p>\n

pSG424<\/span>: \u00a0Expression vector with the SV40 early promoter controlling the GAL4 DNA-binding domain (aa 1-147) (DBD) upstream of the MCS (Sadowski & Ptashne, 1989) <\/strong><\/p>\n

pSG-REL:<\/span> C-terminal sequences of wild-type REL (aa 278-587) fused to GAL4-DBD; a BamHI-KpnI fragment from pBluescript SK+-REL was subcloned into pSG424 digested with BamHI-KpnI (Starczynowski et al, 2003)<\/p>\n

pSG-REL-S484P:<\/span> C-terminal sequences of REL-S484P (aa 278-587) fused to GAL4-DBD; overlapping PCR mutagenesis using pGEM-Hu-cRel as a template was used to mutate REL codon 484 to a Pro codon.\u00a0 The fragment containing REL-S484P was digested with EcoRV-NdeI and subcloned into pSG-REL digested with EcoRV-NdeI (created by J. Leeman, Gilmore lab)<\/p>\n

pSG-REL-S484A:<\/span> C-terminal sequences of REL-S484A (aa 278-587) fused to GAL4-DBD; an EcoRV-NdeI fragment from JD-REL-S484A was subcloned into pSG-REL digested with EcoRV-NdeI<\/p>\n

pSG-REL-S484,494A:<\/span> C-terminal sequences of REL-S484,494A (aa 278-587) fused to GAL4-DBD; an EcoRV-NdeI fragment from JD-REL-S484,494A was subcloned into pSG-REL digested with EcoRV-NdeI<\/p>\n

pSG-REL-S484D,S494A:<\/span> C-terminal sequences of REL-S484D (aa 278-587) fused to GAL4-DBD; overlapping PCR mutagenesis using pSG-REL-S484,494A as a template was used to mutate REL codon 484 to an Asp codon.\u00a0 The fragment containing REL-S484D,S494A was digested with EcoRV-NdeI and subcloned into pSG-REL digested with EcoRV-NdeI<\/p>\n

pSG-REL-S484,S494D:<\/span> C-terminal sequences of REL-S484,494D (aa 278-587) fused to GAL4-DBD; overlapping PCR mutagenesis using pSG-REL-S484D,S494A as a template was used to mutate REL codon 494 to an Asp codon.\u00a0 The fragment containing REL-S484,494D was digested with EcoRV-NdeI and subcloned into pSG-REL digested with EcoRV-NdeI<\/p>\n

pRG424:<\/span> Expression vector with the RSV promoter controlling the GAL4-DBD upstream of the MCS; an NdeI\/Klenow-HindIII fragment from pRSV-bgal was subcloned into pSG424 digested with PvuII-HindIII<\/p>\n

pRG-REL:<\/span> C-terminal sequences of wild-type REL (aa 278-587) fused to GAL4-DBD; an NdeI\/Klenow-HindIII fragment from pRSV-bgal was subcloned into pSG-REL digested with PvuII-HindIII<\/p>\n

pRG-REL-S484,494A:<\/span> C-terminal sequences of REL-S484,494A (aa 278-587) fused to GAL4-DBD; an NdeI\/Klenow-HindIII fragment from pRSV-bgal was subcloned into pSG-REL S484,494A digested with PvuII-HindIII<\/p>\n

pcDNA-GAL4-REL:<\/span> pcDNA containing REL aa 278-587 fused to the GAL4 DBD; a HindIII-XbaI fragment from pSG-REL was subcloned into pcDNA 3.1 (-) digested with HindIII-XbaI (created by J. Leeman, Gilmore lab)<\/p>\n

Vertebrate Reporter Plasmids <\/span> <\/strong><\/p>\n

pRSV-bgal:<\/span> Contains the RSV LTR upstream of the b-galactosidase gene (gift of Douglas Faller, Boston University Medical School)<\/p>\n

PolyA-GAL4E1b GAL4-luc:<\/span> Contains GAL4 DNA-binding sites upstream and a minimal promoter upstream of the luciferase gene (gift of Joseph Lipsick, Stanford Medical School) (Starczynowski et al, 2003)<\/p>\n

pGL3 Promoter Vector:<\/span> a plasmid containing a partial SV40 promoter controlling the luciferase gene (Promega)<\/p>\n

pSV40-Luc:<\/span> Contains the luciferase gene controlled by the SV40 promoter sequences from pSG424; a PvuII-HindIII fragment from pSG424 was subcloned into pGL3 Promoter Vector digested with SmaI-HindIII<\/p>\n

pRSV-Luc:<\/span> Contains the luciferase gene controlled by the RSV promoter sequences from pRSV-bgal; an NdeI\/Klenow-HindIII fragment from pRSV-bgal was subcloned into pGL3 Promoter Vector digested with SmaI-HindIII<\/p>\n

Primers used for PCR <\/span> <\/strong><\/p>\n

SP6 Promoter:<\/span> 5\u2019-GATTTAGGTGACACTATAG-3\u2019<\/p>\n

T7 Promoter:<\/span> 5\u2019-GTAATACGACTCACTATAGGGC-3\u2019<\/p>\n

REL-323-For-EcoRI:<\/span> 5\u2019-CCGGAATTC<\/span>TAGGAGAAGGAAGATACTTC-3\u2019<\/p>\n

REL-FLAG-EcoRI-For: <\/span> 5\u2019-cgaattc<\/span>catggcctccggtgc-3\u2019<\/p>\n

REL-423-Rev-BamHI:<\/span> 5\u2019-CGCAGGATCC<\/span>CAATCATTCCCAACAGG-3\u2019 <\/span><\/p>\n

REL-476-For-EcoRI:<\/span> 5\u2019-CACGAATTC<\/span>GAGAGACTGATAATCCAAGACTTCTG-3\u2019<\/p>\n

REL-504-Rev-HindIII:<\/span> 5\u2019-GCGAAGCTT<\/span>TTAGAGCTGTCTCAAGTCTCTTGGGTCTAAC-3\u2019<\/p>\n

REL-S484P-For:<\/span> 5\u2019-AATCCAAGACTTCTGCC<\/span>CATGAATCTGA-3\u2019<\/p>\n

REL-S484P-Rev:<\/span> 5\u2019-GGTTTTCAAGATTCATGGG<\/span>CAGAAGTCTT-3\u2019<\/p>\n

REL-P484A-For:<\/span> 5\u2019-AATCCAAGACTTCTGGC<\/span>CATGAATCTTG-3\u2019<\/p>\n

REL-P484A-Rev:<\/span> 5\u2019-GGTTTTCAAGATTCATGGC<\/span>CAGAAGTCTTG-3\u2019<\/p>\n

REL-S494A-For:<\/span> 5\u2019-AACCCCTCATGTAATG<\/span>CAGTGTTAGACCC-3\u2019<\/p>\n

REL-S494A-Rev:<\/span> 5\u2019-CTCTTGGGTCTAACACTGC<\/span>ATTACATGAGG-3\u2019<\/p>\n

REL-A484D-For:<\/span> 5\u2019-AATCCAAGACTTCTGGA<\/span>CATGAATCTTG-3\u2019<\/p>\n

REL-A484D-Rev:<\/span> 5\u2019-GGTTTTCAAGATTCATGT<\/span>CCAGAAGTCTTG-3\u2019<\/p>\n

REL-A494D-For:<\/span> 5\u2019-AACCCCTCATGTAATGAC<\/span>GTGTTAGACC-3\u2019<\/p>\n

REL-A494D-Rev:<\/span> 5\u2019-CTCTTGGGTCTAACACGT<\/span>CATTACATGAGG-3\u2019<\/p>\n

Restriction sites and introduced mutations are underlined<\/p>\n

Antisera <\/span><\/strong><\/p>\n

Western blotting <\/strong><\/p>\n

Rabbit anti-REL (C-terminal 15 aa; gift of Nancy Rice):\u00a0 used at 1:10,000 (see Kalaitzidis et al, 2002)<\/p>\n

Immunoprecipitation <\/strong><\/p>\n

Mouse anti-FLAG (Sigma, St. Louis, MO; Catalog #A2220): conjugated to agarose beads<\/p>\n

Mouse anti-NEMO (BD Pharmingen, San Jose, CA; Catalog #559675)<\/p>\n

References<\/span><\/strong> <\/span><\/p>\n

Barkett M, JE Dooher, L Lemonnier, L Simmons, JN Scarpati, Y Wang & TD Gilmore (2001) Three mutations in the retroviral oncoprotein v-Rel render it resistant to cleavage by caspase-3. Biochimica et Biophysica Acta 1526: 25-36<\/p>\n

Gilmore TD, C Cormier, J Jean-Jacques & M-E Gapuzan (2001) Malignant transformation of primary chicken spleen cells by human transcription factor c-Rel. Oncogene 20: 7098-7103<\/p>\n

Kalaitzidis D, RE Davis, A Rosenwald, LM Staudt & TD Gilmore (2002) The human B-cell lymphoma cell line RC-K8 has multiple genetic alterations that dysregulate the Rel\/NF-kB signal transduction pathway. Oncogene 21: 8759-8768<\/p>\n

Leeman JR, MA Weniger, TF Barth & TD Gilmore (2008) Deletion analysis and alternative splicing define a transactivation inhibitory domain in human oncoprotein REL. Oncogene, in revision<\/p>\n

Liang M-C, S Bardhan, C Li, EA Pace, JA Porco Jr & TD Gilmore (2003) Jesterone dimer, a synthetic derivative of the fungal metabolite jesterone, blocks activation of transcription factor nuclear factor kB by inhibiting the inhibitor of kB kinase. Molecular Pharmacology 64: 123-131<\/p>\n

Liang M-C, S Bardhan, EA Pace, D Rosman, JA Beutler, JA Porco Jr & TD Gilmore (2006) Inhibition of transcription factor NF- B signaling proteins IKK and p65 through specific cysteine residues by epoxyquinone A monomer: correlation with its anticancer cell growth activity. Biochemical Pharmacology 71:634-645<\/p>\n

Sadowski I & M Ptashne (1989) A vector for expressing GAL4(1-147) fusions in mammalian cells. Nucleic Acids Research 17: 7539<\/p>\n

Sif S, AJ Capobianco & TD Gilmore (1993) The v-Rel oncoprotein increases expression from Sp1 site-containing promoters in chicken embryo fibroblasts. Oncogene 8: 2501-2509<\/p>\n

Starczynowski DT, JG Reynolds & TD Gilmore (2003) Deletion of either C-terminal transactivation subdomain enhances the in vitro transforming activity of human transcription factor REL in chicken spleen cells. Oncogene 22: 6928-6936<\/p>\n

Starczynowski DT, JG Reynolds & TD Gilmore (2005) Mutations of tumor necrosis factor alpha-responsive serine residues within the C-terminal transactivation domain of human transcription factor REL enhance its in vitro transforming ability. Oncogene 24: 7355-7368<\/p>\n

Starczynowski DT, H Trautmann, C Pott, L Harder, N Arnold, JA Africa, JR Leeman, R Siebert & TD Gilmore (2007) Mutation of an IKK phosphorylation site within the transactivation domain of REL in two patients with B-cell lymphoma enhances REL’s in vitro transforming activity. Oncogene 26: 2685-2694<\/p>\n","protected":false},"excerpt":{"rendered":"

Appendix of Plasmids and Primers for Garbati MR and TD Gilmore (2008) Ser484 and Ser494 in REL are the major sites of IKK phosphorylation in vitro: evidence that IKK does not directly enhance GAL4-REL transactivation. Gene Expression 14: 195-205 pGEM-based Plasmids pGEM4: Cloning vector for in vitro transcription\/translation with either SP6 or T7 promoter elements […]<\/p>\n","protected":false},"author":4258,"featured_media":0,"parent":337,"menu_order":12,"comment_status":"closed","ping_status":"open","template":"","meta":[],"_links":{"self":[{"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/573"}],"collection":[{"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/users\/4258"}],"replies":[{"embeddable":true,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/comments?post=573"}],"version-history":[{"count":0,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/573\/revisions"}],"up":[{"embeddable":true,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/337"}],"wp:attachment":[{"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/media?parent=573"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}