{"id":572,"date":"2011-04-26T15:19:51","date_gmt":"2011-04-26T19:19:51","guid":{"rendered":"https:\/\/www.bu.edu\/nf-kb\/the-gilmore-lab\/data-link-13\/"},"modified":"2011-04-28T19:01:30","modified_gmt":"2011-04-28T23:01:30","slug":"data-link-13","status":"publish","type":"page","link":"https:\/\/www.bu.edu\/nf-kb\/the-gilmore-lab\/data-link-13\/","title":{"rendered":"Data Link 13"},"content":{"rendered":"

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

Garbati MR, G Al\u00e7o and TD Gilmore (2010) Histone acetyltransferase p300 is a coactivator for transcription factor REL and is C-terminally truncated in the human diffuse large B-cell lymphoma cell line RC-K8. Cancer Letters 291: 237-245<\/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) <\/span><\/strong><\/p>\n

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

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

pBS SK+ REL-RHD:<\/span> pBluescript SK+ containing REL aa 305-323; an EcoRV-HindIII fragment from pGEX-5\u2019REL was subcloned into pBluescript SK+ digested with EcoRV-HindIII <\/span><\/strong><\/p>\n

Retroviral Vectors <\/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

<\/span><\/strong><\/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 (Garbati and Gilmore, 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 (Garbati and Gilmore, 2008)<\/p>\n

pcDNA-FLAG-REL-RHD:<\/span> pcDNA containing FLAG-tagged REL aa 1-323; an EcoRV-XhoI fragment from pBS SK+ REL-RHD was subcloned into pcDNA-FLAG-REL digested with EcoRV-XhoI<\/p>\n

pcDNA-FLAG-RELDTAD1:<\/span> pcDNA containing FLAG-tagged RELD424-490<\/span>; an EcoRV-XhoI fragment from pcDNA-RELDTAD1 (Starczynowski et al, 2003) was subcloned into pcDNA-FLAG-REL digested with EcoRV-XhoI (J. Leeman, Gilmore lab)<\/p>\n

pcDNA-FLAG-RELDTAD2:<\/span> pcDNA containing FLAG-tagged RELD58<\/span>; an EcoRV-XhoI fragment from pcDNA-RELD58 (Starczynowski et al, 2003) was subcloned into pcDNA-FLAG-REL digested with EcoRV-XhoI (J. Leeman, Gilmore lab)<\/p>\n

pcDNA-MYC-NEMO:<\/span> pcDNA containing MYC-tagged NEMO (gift of Shigeki Miyamoto, University of Wisconsin, Madison, WI)<\/p>\n

pcDNA-MYC-REL:<\/span> pcDNA containing MYC-tagged REL; a PCR fragment containing REL was digested with EcoRI-XbaI and subcloned into pcDNA-MYC-NEMO digested with EcoRI and XbaI (replacing the NEMO insert with REL)<\/p>\n

pcDNA-MYC-REL-RHD:<\/span> pcDNA containing MYC-tagged REL; an EcoRV-XbaI fragment from pSG-RELD282 (Starczynowski et al.<\/em>, 2003) was subcloned into MYC-REL digested with EcoRV and XbaI<\/p>\n

pCMV-CBP:<\/span> CMV-driven expression vector containing mouse CBP (gift of Ching-Chow Chen (National Taiwan University, Taipei, Taiwan)<\/p>\n

pCMVb-p300-CHA<\/span>:\u00a0 CMV-driven expression vector containing C-terminally HA-tagged human p300 (gift of Myles Brown, MIT)<\/p>\n

pGEX-based GST Fusion Protein Bacterial 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<\/p>\n

pGEX-5\u2019REL: <\/span> REL aa 1-323 fused to GST; a PCR fragment using pGEM-Hu-cRel as a template was digested with EcoRI-BamHI and subcloned into pGEX-KG digested with EcoRI-BamHI (E. Coffee, Dean Tolan lab, Boston University)<\/p>\n

pGEX-3\u2019RELDTAD1:<\/span> RELD424-490 fused to GST; a PCR fragment from pGEM-RELD424-490 (Starczynowski et al.<\/em>, 2003) was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII<\/p>\n

pGEX-3\u2019RELDTAD2:<\/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\u2019RELDTAD1,2:<\/span> REL aa 323-423 fused to GST; a PCR fragment from pGEM-RELD164 (Starczynowski et al.<\/em>, 2003) was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII<\/p>\n

<\/strong><\/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

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

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

SP6 Promoter:\u00a0 5\u2019-GATTTAGGTGACACTATAG-3\u2019<\/p>\n

T7 Promoter:\u00a0 5\u2019-GTAATACGACTCACTATAGGGC-3\u2019<\/p>\n

REL-323-For-EcoRI:\u00a0 5\u2019-CCGGAATTCTAGGAGAAGGAAGATACTTC-3\u2019<\/p>\n

REL-FLAG-EcoRI: \u00a05\u2019-cgaattccatggcctccggtgc-3\u2019<\/p>\n

REL-423-Rev-BamHI:\u00a0\u00a0 5\u2019-CGCAGGATCCCAATCATTCCCAACAGG-3\u2019<\/p>\n

MYC-REL-For-Eco<\/em>RI:\u00a0 5\u2019-CACGAATTCCGCCTCCGGTGCGTATAACC-3\u2019<\/p>\n

MYC-REL-Rev-Xba<\/em>I:\u00a0 5\u2019-GCGTCTAGATTATACTTGAAAAAATTCATATGG-3\u2019<\/p>\n

Restriction sites are underlined.<\/p>\n

Primers used for probe templates and quantitative RT-PCR <\/span> <\/strong><\/p>\n

p300-5\u2019probe-For:\u00a0 5\u2019-CACGAATTCCGGCCTAAACTCTCATCTCC-3\u2019<\/p>\n

p300-5\u2019probe-Rev:\u00a0 5\u2019-CACAAGCTTCATTATCCCTTGTCCATTGC-3\u2019<\/p>\n

p300-exon3-4-For:\u00a0\u00a0\u00a0\u00a0 5\u2019-GAGGAATGCCCAACATGG-3\u2019<\/p>\n

p300-exon11-12-Rev:\u00a0 5\u2019-TGGCCAAATTGATTCAAACC-3\u2019<\/p>\n

p300-exon31-For2:\u00a0 5\u2019-CACGAATTCGGTTGTGCAGCATACCAAGG-3\u2019<\/p>\n

p300-exon31-Rev2:\u00a0 5\u2019-CACGAATTCTGGCGTGAAGGATACTAAGC-3\u2019<\/p>\n

p300-qPCR-exon2-For:\u00a0 5\u2019-AACAGAGCAGTCCTGGATTAGG-3\u2019<\/p>\n

p300-qPCR-exon2-Rev:\u00a0 5\u2019-ATGGCAGGCTGATTTACTGG-3\u2019<\/p>\n

p300-qPCR-exon31-For:\u00a0 5\u2019-GCATATGCTCCCAAATCAGG-3\u2019<\/p>\n

p300-qPCR-exon31-Rev:\u00a0 5\u2019-AACTTGTCTGTGGGGAAACG-3\u2019<\/p>\n

GAPDH-qPCR-For:\u00a0 5\u2019-TGGTATCGTGGAAGGACTCATGAC-3\u2019<\/p>\n

GAPDH-qPCR-Rev:\u00a0 5\u2019-ATGCCAGTGAGCTTCCCGTTCAGC-3\u2019<\/p>\n

Primers used for quantitative PCR of genomic DNA<\/span><\/strong><\/p>\n

p300-GqPCR-exon13-For:\u00a0 5\u2019-CTCGTATGCAACAGCCTTCC-3\u2019<\/p>\n

p300-GqPCR-exon13-Rev:\u00a0 5\u2019-caagaatcaaaaataagtgaaaaatcc-3\u2019<\/p>\n

p300-GqPCR-exon14-For:\u00a0 5\u2019-ATTCACCCTCGCCTGTACC-3\u2019<\/p>\n

p300-GqPCR-exon14-Rev:\u00a0 5\u2019-GGTGTAGGTGTCTGCCTTGG-3\u2019<\/p>\n

p300-GqPCR-exon15-For:\u00a0 5\u2019-gcgtgtgtctcacctacttcc-3\u2019<\/p>\n

p300-GqPCR-exon15-Rev:\u00a0 5\u2019-CTGCTGGTTCTGGTTGATCC-3\u2019<\/p>\n

p300-GqPCR-exon16-For:\u00a0 5\u2019-TACCGAAACAGAAGAGAGAAGC-3\u2019<\/p>\n

p300-GqPCR-exon16-Rev:\u00a0 5\u2019-gctacctccagaacgaatgg-3\u2019<\/p>\n

p300-GqPCR-exon17-For:\u00a0 5\u2019-ACTACGACAGGCACTGATGC-3\u2019<\/p>\n

p300-GqPCR-exon17-Rev:\u00a0 5\u2019-GGGATTCCTAAAAGCTGAGG-3\u2019<\/p>\n

p300-GqPCR-exon18-For: \u00a05\u2019-GGCAGTATGTCGATGATATTTGG-3<\/p>\n

p300-GqPCR-exon18-Rev:\u00a0 5\u2019-TGCCACAACAGTATCCAAGG-3\u2019<\/p>\n

p300-GqPCR-exon19-For:\u00a0 5\u2019-aatggtttcttttgcagTTGG-3\u2019<\/p>\n

p300-GqPCR-exon19-Rev:\u00a0 5\u2019-gcactccctggacatgtgg-3\u2019<\/p>\n

p300-GqPCR-exon20-For:\u00a0 5\u2019-gggctgtgttgtgtgaacg-3\u2019<\/p>\n

p300-GqPCR-exon20-Rev:\u00a0 5\u2019-aatgcagttacttacGTTTGAGG-3\u2019<\/p>\n

p300-GqPCR-exon21-For:\u00a0 5\u2019-gggtgaagtttgttcctttgg-3\u2019<\/p>\n

p300-GqPCR-exon21-Rev:\u00a0 5\u2019-ggatcatctgattggtcatgc-3\u2019<\/p>\n

p300-GqPCR-exon22-For:\u00a0 5\u2019-gctgtctttgtcagaagtcatgg-3\u2019<\/p>\n

p300-GqPCR-exon22-Rev:\u00a0 5\u2019-CAGATGATCTCATGGTGAAGG-3\u2019<\/p>\n

p300-GqPCR-exon23-For:\u00a0 5\u2019-caacggtttatctaagttgtgtaagc-3\u2019<\/p>\n

p300-GqPCR-exon23-Rev:\u00a0 5\u2019-tttggatggtttcttatctaggc-3\u2019<\/p>\n

p300-GqPCR-exon24-For:\u00a0 5\u2019-cctagGGTTGCCATCTACCA-3\u2019<\/p>\n

p300-GqPCR-exon24-Rev:\u00a0 5\u2019-tttggatccacgaggagaag-3\u2019<\/p>\n

p300-GqPCR-exon25-For:\u00a0 5\u2019-TGTGGACAGTGGAGAGATGG-3\u2019<\/p>\n

p300-GqPCR-exon25-Rev:\u00a0 5\u2019-GAGGGCAGTCAGAGCCATAC-3<\/p>\n

p300-GqPCR-exon27-For:\u00a0 5\u2019-TTACACAACAGGGCATATTTGG-3\u2019<\/p>\n

p300-GqPCR-exon27-Rev:\u00a0 5\u2019-CTTGTAGTCATGGACAATACGC-3<\/p>\n

p300-qPCR-exon31-For:\u00a0 5\u2019-GCATATGCTCCCAAATCAGG-3\u2019<\/p>\n

p300-qPCR-exon31-Rev:\u00a0 5\u2019-AACTTGTCTGTGGGGAAACG-3\u2019<\/p>\n

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

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

rabbit anti-REL (C-terminal 15 aa, #265, Nancy Rice, NCI, Frederick, MD) used at 1:10,000<\/p>\n

rabbit anti-REL (NLS, #1206, Nancy Rice, NCI) used at 1:2500<\/p>\n

rabbit anti-CBP (#4772, Cell Signaling Technology, Danvers, MA) used at 1:1000<\/p>\n

mouse anti-CBP (sc-7300, Santa Cruz Biotechnology, Santa Cruz, CA) used at 1:200<\/p>\n

rabbit anti-p300 (N-terminal, sc-584, Santa Cruz Biotechnology) used at 1:200<\/p>\n

rabbit anti-p300 (C-terminal, sc585, Santa Cruz Biotechnology) used at 1:200<\/p>\n

rabbit anti-IkBa (sc-371, Santa Cruz Biotechnology) used at 1:500<\/p>\n

rabbit b-tubulin (sc-9104, Santa Cruz Biotechnology) used at 1:500<\/p>\n

rabbit anti-HA (sc-805, Santa Cruz Biotechnology) used at 1:500<\/p>\n

rabbit anti-FLAG (#2368, Cell Signaling Technology) used at 1:1000<\/p>\n

mouse anti-MYC (sc-40, Santa Cruz Biotechnology) used at 1:200<\/p>\n

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

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

mouse anti-FLAG (#A2220, Sigma, St. Louis, MO) conjugated to agarose beads; used 40 ml\/ml<\/p>\n

rabbit anti-p300 (N-terminal, sc-584, Santa Cruz Biotechnology) used 6 mg\/ml<\/p>\n

rabbit normal IgG (sc-2027, Santa Cruz Biotechnology) used 6 mg\/ml<\/p>\n

mouse anti-MYC (sc-40, Santa Cruz Biotechnology) used 6 mg\/ml<\/p>\n

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

Barkett M, JE Dooher, L Lemonnier, L Simmons, JN Scarpati, Y Wang and 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

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

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

Leeman JR, MA Weniger, TF Barth and TD Gilmore (2008) Deletion analysis and alternative splicing define a transactivation inhibitory domain in human oncoprotein REL. Oncogene 27: 6770-6781<\/p>\n

Mitchell T and B Sugden (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

Sif S, AJ Capobianco and 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 and 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 of Plasmids and Primers for Garbati MR, G Al\u00e7o and TD Gilmore (2010) Histone acetyltransferase p300 is a coactivator for transcription factor REL and is C-terminally truncated in the human diffuse large B-cell lymphoma cell line RC-K8. Cancer Letters 291: 237-245 pGEM-based Plasmids pGEM4: Cloning vector for in vitro transcription\/translation with either SP6 or […]<\/p>\n","protected":false},"author":4258,"featured_media":0,"parent":337,"menu_order":11,"comment_status":"closed","ping_status":"open","template":"","meta":[],"_links":{"self":[{"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/572"}],"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=572"}],"version-history":[{"count":3,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/572\/revisions"}],"predecessor-version":[{"id":618,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/572\/revisions\/618"}],"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=572"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}