{"id":581,"date":"2011-04-26T15:24:57","date_gmt":"2011-04-26T19:24:57","guid":{"rendered":"https:\/\/www.bu.edu\/nf-kb\/?page_id=581"},"modified":"2011-04-30T14:02:13","modified_gmt":"2011-04-30T18:02:13","slug":"data-link-8","status":"publish","type":"page","link":"https:\/\/www.bu.edu\/nf-kb\/the-gilmore-lab\/data-link-8\/","title":{"rendered":"Data Link 8"},"content":{"rendered":"

APPENDIX <\/strong><\/h3>\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 can enhance its in vitro transforming ability. Oncogene 24: 7355-7368<\/p>\n

Vectors for In <\/em><\/strong>V<\/em><\/strong>itro<\/em> Transcription\/Translation <\/strong><\/p>\n

pGEM4: <\/span> Vector for in vitro transcription\/translation (Promega)<\/p>\n

pGEM-Hu-cRel:<\/span> Vector for in vitro transcription\/translation of wild-type human REL using SP6 polymerase; XbaI-XhoI\/Klenow fragment of REL from CM216 subcloned into XbaI-HincII-digested pGEM4 (Barkett et al., 2001) <\/span><\/p>\n

pGEM-RELD424-490:<\/span> aa 424-490 deleted from within the C terminal half of REL; an EcoRV-XhoI fragment from a PCR-generated deletion (ask for details for primers) was used to replace an EcoRV-XhoI fragment in pGEM-Hu-c-Rel<\/p>\n

pGEM-RELD424-490\/bgal<\/span>: aa 424-490 from RELwere replaced with 66 aa of b-galactosidase sequences from pBluescript SK+ (ask for details for primers)<\/p>\n

pGEM-REL-S454A<\/span>: pBluescriptSK+-REL-S454A digested with SwaI was subcloned into pGEM-RELD164 (SwaI) (Starczynowski et al, 2003)<\/p>\n

pGEM-REL-S460A<\/span>: pBluescriptSK+-REL-S460A digested with SwaI-XhoI was subcloned into pGEM-RELD150 (SwaI-XhoI) (Starczynowski et al, 2003)<\/p>\n

pGEM-REL-S471A<\/span>:\u00a0\u00a0 PCR generated fragment (see Table 2.1 for primers) using pGEM-Hu-c-Rel as a template was subcloned EcoRV-XhoI into pGEM-RELD424-490 (EcoRV-XhoI)<\/p>\n

pGEM-REL-S491,494A<\/span>: pBluescriptSK+-REL-S491,494A digested with SwaI was subcloned into pGEM-RELD164 (SwaI) (Starczynowski et al, 2003)<\/p>\n

Retroviral vectors<\/h3>\n

JD-REL:<\/span> JD214BS+ containing human REL<\/em> subcloned as an XbaI-XhoI fragment into JD214BS+ digested with XbaI-SalI (Gilmore et al, 2001) <\/span><\/p>\n

JD-RELD424-490:<\/span> Spleen necrosis virus vector for expressing RELD424-490; JD214BS+ containing human RELD424-490 subcloned as an XbaI-XhoI fragment into JD214BS+ digested with XbaI-SalI\u00a0 <\/span><\/p>\n

JD-RELD424-490\/bgal<\/span>:\u00a0 Spleen necrosis virus vector for expressing RELD424-490\/bgal; JD214BS+ containing RELD424-490\/bgal, subcloned as an XbaI-NdeI fragment into JD-RELD424-490 digested with XbaI-NdeI (Starczynowski et al, 2003) <\/span><\/p>\n

JD-REL-S443,444,447A<\/span>: JD214BS+ containing REL-S443,444,447A, subcloned as an ApaI-NdeI\u00a0 fragment from pUC18-REL-S443,444,447A into JD-RELD424-490 digested with ApaI-NdeI <\/span><\/p>\n

JD-REL-S454A<\/span>:\u00a0\u00a0 JD214BS+ containing REL-S454A, subcloned as an Xba-XhoI fragment from pGEM-REL-S454A into JD214BS+ digested with XbaI-SalI<\/p>\n

JD-REL-S460A<\/span>:\u00a0 JD214BS+ containing REL-S460A, subcloned as an Xba-XhoI fragment from pGEM-REL-S460A into JD214BS+ digested with XbaI-SalI<\/p>\n

JD-REL-S471N<\/span>:\u00a0\u00a0 JD214BS+ containing REL-S471N, subcloned as an ApaI-HindIII fragment from pGEM-REL-S471N into JD-RELD424-490 digested with ApaI-HindIII<\/p>\n

JD-REL-S471A<\/span>: JD214BS+ containing REL-S471D, subcloned as an ApaI-HindIII fragment from a PCR-generated fragment into JD-RELD424-490\u00a0 digested with ApaI-HindIII<\/p>\n

JD-REL-S471D<\/span>:\u00a0\u00a0 JD214BS+ containing REL-S471D, subcloned as an Xba-HindIII fragment from pGEM-REL-S471D into JD214 BS+ digested with XbaI-HindIII<\/p>\n

JD-REL-S491,494A<\/span>:\u00a0 JD214BS+ containing REL-S491,494A, subcloned as an Xba-XhoI fragment from pGEM-REL-S494,494A into JD214 BS+ digested with XbaI-SalI<\/p>\n

JD-REL-S533,536A<\/span>: JD214BS+ containing REL-S533,536A, subcloned as an ApaI-NdeI fragment from pUC18-REL-S533,536A into JD-REL-D424-490 digested with ApaI-NdeI<\/p>\n

JD-REL-S460F<\/span>:\u00a0 JD214BS+ containing REL-S460F, subcloned as an Xba-XhoI fragment from pGEM-REL-S460F into JD214BS+ digested with XbaI-SalI<\/p>\n

Cloning Vectors<\/h3>\n

pBluescript SK+:<\/span> Plasmid containing the MCS within lacZ<\/em> (Stratagene) <\/span><\/p>\n

pBluescript SK+-REL:<\/span> Wild-type REL<\/em> fragment (EcoRV-XhoI) subcloned into the corresponding sites (EcoRV-XhoI) of pBluescript SK+\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/span><\/p>\n

pBluescript SK+-RELD424-490:<\/span> RELD424-490 EcoRV-XhoI fragment subcloned into EcoRV-XhoI sites of pBluescript SK+ <\/span><\/p>\n

pBluescriptSK+-3\u2019REL-S454A<\/span>: Plasmid containing REL fragment from SwaI to 5\u2019 end; REL-S454A fragment (SacI-BglII) from pUC18-3\u2019REL-S454A subcloned into pBluescript SK+ (SacI-BamHI)<\/p>\n

pBluescriptSK+-3\u2019REL-S460A<\/span>: Plasmid containing REL fragment from SwaI to 5\u2019 end; REL-S460A fragment (SacI-BglII) from pUC18-3\u2019REL-S454A subcloned into pBluescript SK+ (SacI-BamHI)<\/p>\n

pBluescriptSK+-3\u2019REL-S491,494A<\/span>: Plasmid containing REL fragment from SwaI to 5\u2019end; REL-S491,494A fragment (SacI-BglII) from pUC18-3\u2019REL-S454A subcloned into pBluescript SK+ (SacI-BamHI)<\/p>\n

pBluescriptSK+-3\u2019REL-S454A<\/span>: Plasmid containing REL fragment from EcoRV to 5\u2019 end; REL-S454A fragment (EcoRV-HindIII) from JD-REL-S454A subcloned into the corresponding sites (EcoRV-HindIII) of pBluescript SK+<\/p>\n

pBluescriptSK+-3\u2019REL-S460A<\/span>: Plasmid containing REL fragment from EcoRV to 5\u2019 end; REL-S460A fragment (EcoRV-HindIII) from JD-REL-S460A subcloned into the corresponding sites (EcoRV-HindIII) of pBluescript SK+<\/p>\n

pBluescriptSK+-3\u2019REL-S471N<\/span>: Plasmid containing REL fragment from EcoRV to 5\u2019 end; REL-S471N fragment (EcoRV-HindIII) from JD-REL-S471N subcloned into the corresponding sites (EcoRV-HindIII) of pBluescript KS+<\/p>\n

pBluescriptSK+-3\u2019REL-S471A<\/span>: Plasmid containing REL fragment from EcoRV to 5\u2019 end; REL-S471A fragment (EcoRV-HindIII) from JD-REL-S471A subcloned into the corresponding sites (EcoRV-HindIII) of pBluescript SK+<\/p>\n

pBluescriptSK+-3\u2019REL-S471D<\/span>: Plasmid containing REL fragment from EcoRV to 5\u2019 end; REL-S471D fragment (EcoRV-HindIII) from JD-REL-S471D subcloned into the corresponding sites (EcoRV-HindIII) of pBluescript SK+<\/p>\n

pBluescriptSK+-3\u2019REL-S491,494A<\/span>: Plasmid containing REL fragment from EcoRV to 5\u2019 end; REL-S491,494A fragment (EcoRV-HindIII) from JD-REL-S491,494A subcloned into the corresponding sites (EcoRV-HindIII) of pBluescript SK+<\/p>\n

pBluescriptSK+-3\u2019REL-S460F<\/span>: Plasmid containing REL fragment from EcoRV to 5\u2019end; REL-S460F fragment (EcoRV-HindIII) from JD-REL-S460F subcloned into the corresponding sites (EcoRV-HindIII) of pBluescript SK+<\/p>\n

Expression Vectors<\/h3>\n

SG424: <\/span> Expression plasmid containing GAL4 DNA-binding domain (DBD) upstream of MCS and downstream of SV40 promoter (Sadowski and Ptashne, 1989) <\/span><\/p>\n

SG-3\u2019REL:<\/span> Wild-type human c-Rel (aa 278-587) fused to GAL4 DBD; pBluescript SK+-REL cut with BamHI-KpnI was subcloned into BamHI-KpnI sites in SG424 <\/span><\/p>\n

SG-3\u2019RELD424-490:<\/span> REL (aa 278-423,491-587) fused to GAL4 DBD pBluescript SK+RELD424-490\u00a0 cut with BamHI-KpnI was subcloned into BamHI-KpnI sites in SG424 <\/span><\/p>\n

SG-3\u2019REL-S443,444,447A<\/span>: REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S443,444,447A cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424<\/p>\n

SG-3\u2019REL-S454A<\/span>:\u00a0\u00a0\u00a0 REL mutant fused to GAL4 DBD; pBluescript SK+-REL-454A cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424<\/p>\n

SG-3\u2019REL-S460A<\/span>:\u00a0 REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S460A cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424<\/p>\n

SG-3\u2019REL-S471A<\/span>:\u00a0 REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S471A cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424<\/p>\n

SG-3\u2019REL-S471N<\/span>:\u00a0 REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S471N cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424<\/p>\n

SG-3\u2019REL-S471D<\/span>:\u00a0 REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S471D cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424<\/p>\n

SG-3\u2019REL-S491,494A<\/span>: REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S491,494A cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424<\/p>\n

SG-3\u2019REL-S533,536A<\/span>: REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S533,536A cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424<\/p>\n

SG-3\u2019REL-S460F<\/span>:\u00a0\u00a0 REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S460F cut with BamHI-KpnI was subcloned into BamHI-KpnI in pSG424<\/p>\n

CM216:<\/span> CMV promoter expression vector for full-length REL (Inder Verma)<\/p>\n

pcDNA3.1(-):<\/span> CMV promoter expression vector (Promega) <\/span><\/p>\n

pcREL-S443,444,447A<\/span>: pcDNA3.1(-) containing REL-S443,444,447A, subcloned as an XbaI-HindIII fragment from JD-REL-S443,444,447A into pcDNA3.1(-) digested with XbaI-HindIII<\/p>\n

pcREL-S454A<\/span>:\u00a0\u00a0 pcDNA3.1(-) containing REL-S454A. REL-S454A subcloned as an XbaI-HindIII fragment from JD-REL-S454A into pcDNA3.1(-) digested with XbaI-HindIII<\/p>\n

pcREL-S460A<\/span>:\u00a0 pcDNA3.1(-) containing REL-S460A. REL-S460A subcloned as an XbaI-HindIII fragment from JD-REL-S460A into pcDNA3.1(-) digested with XbaI-HindIII<\/p>\n

pcREL-S471A<\/span>:\u00a0\u00a0\u00a0 pcDNA3.1(-) containing REL-S471A. REL-S471A subcloned as an XbaI-HindIII fragment from JD-REL-S471A into pcDNA3.1(-) digested with XbaI-HindIII<\/p>\n

pcREL-S471N<\/span>:\u00a0 pcDNA3.1(-) containing REL-S471N. REL-S471N subcloned as an XbaI-HindIII fragment from JD-REL-S471N into pcDNA3.1(-) digested with XbaI-HindIII<\/p>\n

pcREL-S471D<\/span>:\u00a0 pcDNA3.1(-) containing REL-S471D. REL-S471D subcloned as an XbaI-HindIII fragment from JD-REL-S471D into pcDNA3.1(-) digested with XbaI-HindIII<\/p>\n

pcREL-S491,494A<\/span>:\u00a0 pcDNA3.1(-) containing REL-S491,494A. REL-S491,494A subcloned as an XbaI- HindIII fragment from JD-REL-S491,494A into pcDNA3.1(-) digested with XbaI-HindIII<\/p>\n

pcREL-S533,536A:<\/span> pcDNA3.1(-) containing REL-S533,536A. REL-S533,536A subcloned as an XbaI-HindIII fragment from JD-REL-S533,536A into pcDNA3.1(-) digested with XbaI-HindIII<\/p>\n

pcREL-S460F:<\/span> pcDNA3.1(-) containing REL-S460F. REL-S460F subcloned as an XbaI-HindIII fragment from JD-REL-S460F into pcDNA3.1(-) digested with XbaI-HindIII<\/p>\n

pcRELD424-490<\/span>:\u00a0 pcDNA3.1(-) containing RELD424-490. RELD424-490 subcloned as an XbaI-HindIII fragment from JD-RELD424-490 into pcDNA3.1(-) digested with XbaI-HindIII<\/p>\n

pUC18-3\u2019REL-S443,444,447A<\/span>: REL-S443,444,447A (aa 309-587) fused to GAL4 DBD (Martin et al, 2001)<\/p>\n

pUC18-3\u2019REL-S454A<\/span>: REL-S454A (aa 309-587) fused to GAL4 DBD (Martin et al, 2001)<\/p>\n

pUC18-3\u2019REL-S460A<\/span>: REL-S460A (aa 309-587) fused to GAL4 DBD (Martin et al, 2001)<\/p>\n

pUC18-3\u2019REL-S491,494A<\/span>: REL-S491,494A (aa 309-587) fused to GAL4 DBD (Martin et al, 2001)<\/p>\n

pUC18-3\u2019REL-S533,536A<\/span>: REL-S533,536A (aa 309-587) fused to GAL4 DBD (Martin et al, 2001)<\/p>\n

pRc\/CMV-REL-S471N<\/span>: CMV-containing plasmid for expression of REL-S471N (Martin et al, 2000)<\/p>\n

Reporter Plasmids<\/h3>\n

3x-kB-pGL2:<\/span> 3x-kB-Luciferase-pGL2 reporter plasmid has a minimal c-fos<\/em> promoter element and three copies of the major histocompatibility complex (MHC) class I kB element (TGGGGATTCCCCA) upstream of Luciferase (Mitchell and Sugden, 1995) .<\/p>\n

IkB-pGL2:<\/span> IkBa-Luciferase-pGL2 reporter plasmid contains a 1.3 kb Hin<\/em>dIII-Eco<\/em>RI fragment of the chicken IkBa genomic clone, containing the transcriptional start site and 900 bp of upstream sequence upstream of Luciferase (Schatzle et al, 1995) .<\/p>\n

SOD2-pGL3:<\/span> The SOD2-Luciferase-pGL3 reporter plasmid contains 3.3 kb of the human SOD2<\/em> gene promoter and 0.4 kb of the SOD2<\/em> intronic enhancer upstream of Luciferase (Abid et al, 2004) .<\/p>\n

CMV-bgal<\/span>:\u00a0 CMV promoter\u2013driven expression plasmid for b-galactosidase; used for normalization of transfection efficiency<\/p>\n

pGK-bgal<\/span>:\u00a0 SV40 promoter-driven expression plasmid for b-galactosidase; used for normalization of transfection efficiency<\/p>\n

Primers used for PCR<\/h3>\n

REL Forward<\/span>:\u00a0\u00a0\u00a0 5\u2019-GAAGTTAGTGAATCTATCGATTTT-3\u2019<\/p>\n

REL Reverse<\/span>:\u00a0\u00a0\u00a0\u00a0 5\u2019-CAATTGACTGACTCCTTTCCATATGAA-3\u2019<\/p>\n

REL-S471A Forward<\/span>: 5\u2019-ATGACAACCAGCGCT<\/span>GACAGCATGGGAGAG-3\u2019<\/p>\n

REL-S471A Reverse:<\/span> 5\u2019-CTCTCCCATGCTGTCAGC<\/span>GCTGGTTGTCAT-3\u2019<\/p>\n

REL-S471D Forward<\/span>: 5\u2019-ATGACAACCAGCGAT<\/span>GACAGCATGGGAGAG-3\u2019<\/p>\n

REL-S471D Reverse<\/span>:\u00a0 5\u2019-CTCTCCCATGCTGTCATC<\/span>GCTGGTTGTCAT-3\u2019<\/p>\n

Oligonucleotide used for kB probe (annealed to be double-stranded)<\/h3>\n

kB-WT: <\/span> 5\u2019-TCGAGAGGTCGGGAAATTCC<\/span>CCCCCG-3\u2019kB site underlined<\/p>\n

References<\/strong><\/h3>\n

Abid MR, IG Schoots, KC Spokes, SQ Wu, Mawhinney C & WC Aird (2004) Vascular endothelial growth factor-mediated induction of manganese superoxide dismutase occurs through redox-dependent regulation of forkhead and IkB\/NF-kB. Journal of Biological Chemistry 279: 44030-44038<\/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

Martin AG, B San-Antonio & M Fresno (2001) Regulation of NF-kB transactivation: implication of phosphatiylinositol 3-kinase and protein kinase C zeta in c-Rel activation by tumor necrosis factor alpha. Journal of Biological Chemistry 276: 15840-15849<\/p>\n

Mitchell T & Sugden B (1995) Stimulation of NF-kB-mediated transcription by mutant derivatives of the latent membrane protein of Epstein-Barr virus. Journal of Virology 69: 2968-2976<\/p>\n

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

Schatzle JD, J Kralova & HR Bose Jr (1995) Avian IkBa is transcriptionally induced by c-Rel and v-Rel with different kinetics. Journal of Virology 69: 5383-5390<\/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","protected":false},"excerpt":{"rendered":"

APPENDIX 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 can enhance its in vitro transforming ability. Oncogene 24: 7355-7368 Vectors for In Vitro Transcription\/Translation pGEM4: Vector for in vitro transcription\/translation (Promega) pGEM-Hu-cRel: Vector for in vitro transcription\/translation […]<\/p>\n","protected":false},"author":4258,"featured_media":0,"parent":337,"menu_order":16,"comment_status":"closed","ping_status":"open","template":"","meta":[],"_links":{"self":[{"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/581"}],"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=581"}],"version-history":[{"count":8,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/581\/revisions"}],"predecessor-version":[{"id":604,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/581\/revisions\/604"}],"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=581"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}