{"id":560,"date":"2011-04-26T15:11:07","date_gmt":"2011-04-26T19:11:07","guid":{"rendered":"https:\/\/www.bu.edu\/nf-kb\/?page_id=560"},"modified":"2011-04-28T18:54:19","modified_gmt":"2011-04-28T22:54:19","slug":"data-link","status":"publish","type":"page","link":"https:\/\/www.bu.edu\/nf-kb\/the-gilmore-lab\/data-link\/","title":{"rendered":"Data Link"},"content":{"rendered":"

Appendix<\/span><\/strong><\/h2>\n

Descriptions of plasmids used to express the Rel proteins studied.<\/span> The source of the plasmid is indicated in parentheses.<\/span><\/h2>\n

BC102<\/span>:\u00a0 Vector alone (Kamens & Brent, 1991)<\/p>\n

BC102-Spe\/Sna<\/span>:\u00a0 BC102 with linker containing SpeI and SnaBI sites at the unique EcoRI site (Dave & Gilmore, unpublished)<\/p>\n

JK v-Rel<\/span>: Wild-type full-length v-Rel in BC102 (Kamens & Brent, 1991)<\/p>\n

JK c-Rel<\/span>:\u00a0 Wild-type full-length c-Rel in BC102 (Kamens & Brent, 1991)<\/p>\n

BC 5\u2019v-Rel<\/span>:\u00a0\u00a0\u00a0 Contains sequences up to v-rel<\/em>\u2019s unique HincII restriction site, includes mainly the N-terminal Rel Homology domain and lacking the C-terminal transactivation domains; from amino acids (aa) 1-331 (Mosialos & Gilmore, 1993)<\/p>\n

JK 5\u2019c-Rel<\/span>: Contains sequences up to c-rel<\/em>\u2019s unique HincII restriction site, including mainly the N-terminal Rel Homology domain and lacking C-terminal transactivation domains; from aa 1-322 (Harkness & Gilmore, unpublished)<\/p>\n

BC 5\u2019V\/C<\/span>: A 5\u2019Rel protein that consists of N-terminal 5\u2019v-Rel sequences up to the ClaI restriction site (aa 148) and C-terminal 5\u2019c-Rel sequences to the Hinc II site (Harkness & Gilmore, unpublished)<\/p>\n

BC 5\u2019C\/V<\/span>: A 5\u2019Rel protein that consists of N-terminal 5\u2019c-Rel sequences up to the ClaI restriction site and C-terminal 5\u2019v-Rel sequences to the HincII site (Harkness & Gilmore, unpublished)<\/p>\n

BC 5′D<\/span>Env-v-Rel :<\/span> A 5’v-Rel protein that consists of a deletion of aa 1-11 (Envelope sequence) and the addition of an AUG start codon (this publication) BC 5’Env-c-Rel:<\/span> A 5’c-Rel protein that consists of an addition of the 11 aa Envelope sequence (this publication)<\/p>\n

BC-p100<\/span>:\u00a0\u00a0 Human NF-kB1-p100 subcloned into BC102<\/p>\n

BC-p52<\/span>:\u00a0 Human NF-kB-p52 (aa 1-399, up to an internal XmaI site) subcloned into BC102<\/p>\n

BC-p85<\/span>:\u00a0\u00a0 Human NF-kB1-p85 cDNA from Hut-78 cells subcloned into BC102<\/p>\n

\n

Descriptions of Plasmids used to express the GAL4-Rel proteins.<\/span><\/p>\n

The source of the plasmid is indicated in parentheses.<\/span><\/p>\n

GBT9<\/span>: GAL4 DNA-binding domain alone, aa 1-147 (Clontech)<\/p>\n

GB 5\u2019v-Rel<\/span>: 5\u2019v-Rel from aa 2-331 fused to GAL4 DNA-binding domain (Harkness & Gilmore, unpublished)<\/p>\n

GB 5\u2019c-Rel<\/span>: 5\u2019c-Rel from aa 1-322 fused to GAL4 DNA-binding domain (Harkness & Gilmore, unpublished)<\/p>\n

GB 3\u2019v-Rel<\/span>: GAL4 DNA-binding domain fused to carboxy-terminal v-Rel sequences (aa 332-503) starting from the Hinc II site, includes v-Rel transactivation domains (Koedood & Gilmore, unpublished)<\/p>\n

GB 3\u2019c-Rel<\/span>: GAL4 DNA-binding domain fused to C-terminal c-Rel sequences (aa 324-598) starting from Hinc II site, includes c-Rel transactivation domains (Gilmore, unpublished)<\/p>\n

GB 5\u2019v-Rel SPW<\/span>: GB 5\u2019v-Rel with a proline-tryptophan insertion mutation in the Rel Homology domain at aa 273 that inhibits the formation of homodimers (Harkness & Gilmore, unpublished)
\nGB 5’V\/C (Cla)<\/span>: A GAL4-5\u2019Rel protein which consists of N-terminal 5\u2019v-Rel sequences up to Cla I restriction site and C-terminal 5\u2019c-Rel sequences to the Hinc II site (this publication)<\/p>\n

GB 5’C\/V (Cla):<\/span> A GAL4-5\u2019Rel protein which consists of N-terminal 5\u2019c-Rel sequences up to Cla I restriction site and C-terminal 5\u2019v-Rel sequences to the Hinc II site (this publication)
\nGB 5’V\/C (Sca)<\/span>: A GAL4-5\u2019Rel protein which consists of N-terminal 5\u2019v-Rel sequences up to Sca I restriction site and C-terminal 5\u2019c-Rel sequences to the Hinc II site (this publication)<\/p>\n

GB 5’C\/V (Sca):<\/span> A GAL4-5\u2019Rel protein which consists of N-terminal 5\u2019c-Rel sequences up to Sca I restriction site and C-terminal 5\u2019v-Rel sequences to the Hinc II site (this publication)<\/p>\n

GB 5′D<\/span>Env-v-Rel :<\/span> A GAL4-5’v-Rel protein which consists of a deletion of aa 1-11, v-Rel Envelope sequence (this publication)<\/p>\n

GB 5’Env-c-Rel:<\/span> A GAL4-5’c-Rel protein which consists of an addition of the 11 aa
\nv-Rel Envelope sequence (this publication)<\/p>\n

GB 5’Env C\/V (Cla)<\/span>: A GAL4-5’CV (Cla) protein with the addition of the 11 aa
\nv-Rel Envelope sequence at its N terminus (this publication)<\/p>\n

GB 5’RevA-v-Rel:<\/span> A GAL4-5’v-Rel protein with a substitution of its Envelope sequence for Rev-A Envelope sequence (this publication)<\/p>\n

GB 5’RevA-c-Rel<\/span>: A GAL4-5’c-Rel protein which consists of an addition of the 11 aa Rev-A Envelope sequence (this publication)<\/p>\n

GB 5’Env:<\/span> A fusion that only consists of the GAL4 DNA-binding domain fused to the 11 Envelope-derived aa (this publication)<\/p>\n

GB-3’p100<\/span>:\u00a0 A fusion that contains aa 399-899 of human p100 (from XmaI site at codon 399)<\/p>\n

GB-3’p85<\/span>:\u00a0 A fusion that contains aa 399-666 from human p85 (from XmaI site at codon 399)<\/p>\n

\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Construction of BC102 and GBT9 (GAL4) subclones.<\/span><\/span><\/td>\n <\/span><\/td>\n <\/span><\/td>\n<\/tr>\n
Subclone<\/span><\/strong><\/td>\nOriginal Plasmid containing rel<\/em> fragment and restriction site (insert)<\/span><\/strong><\/td>\nVector and restriction site cloned into<\/span><\/strong><\/td>\n<\/tr>\n
<\/span><\/td>\n <\/span><\/td>\n <\/span><\/td>\n<\/tr>\n
BC 5’v-Rel <\/span>D<\/span>Env <\/span><\/td>\nGB 5’v-Rel <\/span>D<\/span>Env (Sma I-Hinc II)<\/span><\/td>\nBC102 (SnaB I)<\/span><\/td>\n<\/tr>\n
<\/span><\/td>\n <\/span><\/td>\n <\/span><\/td>\n<\/tr>\n
BC 5’Env-c-Rel <\/span><\/td>\nPCR fragment (Spe I-SnaB I)<\/span><\/td>\nBC102 (Spe I-SnaB I)<\/span><\/td>\n<\/tr>\n
<\/span><\/td>\n <\/span><\/td>\n <\/span><\/td>\n<\/tr>\n
GB 5’V\/C (Cla) <\/span><\/td>\nGB 5’v-Rel (Cla I-Sal I)<\/span><\/td>\nGB 5’c-Rel (Cla I-Sal I)<\/span><\/td>\n<\/tr>\n
<\/span><\/td>\n <\/span><\/td>\n <\/span><\/td>\n<\/tr>\n
GB 5’C\/V (Cla) <\/span><\/td>\nGB 5’c-Rel (Cla I-Sal I)<\/span><\/td>\nGB 5’v-Rel (Cla I-Sal I)<\/span><\/td>\n<\/tr>\n
<\/span><\/td>\n <\/span><\/td>\n <\/span><\/td>\n<\/tr>\n
GB 5’V\/C (Sca) <\/span><\/td>\nGB 5’v-Rel (Sca I-partial Sal I)<\/span><\/td>\nGB 5’c-Rel (Sca I-Sal I)<\/span><\/td>\n<\/tr>\n
<\/span><\/td>\n <\/span><\/td>\n <\/span><\/td>\n<\/tr>\n
GB 5’C\/V (Sca) <\/span><\/td>\nGB 5’c-Rel (Sca I-partial Sal I)<\/span><\/td>\nGB 5’v-Rel (Sca I-Sal I)<\/span><\/td>\n<\/tr>\n
<\/span><\/td>\n <\/span><\/td>\n <\/span><\/td>\n<\/tr>\n
GB 5′<\/span>D<\/span>Env-v-Rel <\/span><\/td>\nPCR fragment (Sal I-Ava I)<\/span><\/td>\nGBT9 (SalI)<\/span><\/td>\n<\/tr>\n
<\/span><\/td>\n <\/span><\/td>\n <\/span><\/td>\n<\/tr>\n
GB 5’Env-c-Rel <\/span><\/td>\nGB 5’V\/C (Sca I-HincII)<\/span><\/td>\nGBT9 (Sca I\/HincII)<\/span><\/td>\n<\/tr>\n
<\/span><\/td>\n <\/span><\/td>\n <\/span><\/td>\n<\/tr>\n
GB Env<\/span><\/td>\nEnv oligo (EcoR I-Sma I)<\/span><\/td>\nGBT9 (EcoR I-Sma I)<\/span><\/td>\n<\/tr>\n
<\/span><\/td>\n <\/span><\/td>\n <\/span><\/td>\n<\/tr>\n
GB 5’Env C\/V(Cla) <\/span><\/td>\nGB 5’Env c-Rel-(Cla I-Sal I)<\/span><\/td>\nGB 5’v-Rel (Cla I-Sal I)<\/span><\/td>\n<\/tr>\n
<\/span><\/td>\n <\/span><\/td>\n <\/span><\/td>\n<\/tr>\n
GB 5’RevA Env-v-Rel <\/span><\/td>\nPCR fragment (Xma I-Hinc II)<\/span><\/td>\nGBT9 (Xma I\/Pst I + T4 polmerase)<\/span><\/td>\n<\/tr>\n
<\/span><\/td>\n <\/span><\/td>\n <\/span><\/td>\n<\/tr>\n
GB 5’RevA Env-c-Rel <\/span><\/td>\nPCR fragment (Xma I-Hinc II)<\/span><\/td>\nGBT9 (Xma I\/Pst I + T4 polmerase)<\/span><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n
\n

Oligonucleotide sequences used as primers in PCR and subclone construction<\/span><\/p>\n

Sequence of Oligonucleotides Used to Create Plasmids by PCR<\/span><\/span><\/strong><\/p>\n

BC 5’Env-c-Rel<\/p>\n

Primer 1: <\/span>GA CTA GTC ATG GAC TTT CTC ACC AAC CTC CGA TTC<\/span><\/p>\n

Primer 2: <\/span>CTA TAC GTA CTA GTT GAC AGT GGG GAT TGG AGC TGC TTT TGG<\/span><\/p>\n

GB 5′D<\/span>Env-v-Rel<\/span><\/p>\n

Primer 1: <\/span>C CCT CGA GGG ATG GGT ATC TCA GAG CCC TAC ATT<\/span><\/p>\n

Primer 2: T7 primer<\/span><\/p>\n

GB 5’RevA-v-Rel & 5’RevA-c-Rel<\/span><\/p>\n

Primer 1: <\/span>C CCC CCG GGG ATG GAC TGT CTC ACC GAC CTC CGA TCC ACT GAG<\/span><\/p>\n

Primer 2: <\/span>CTA TAC GTA CTA GTT GAC AGT GGG GAT TGG AGC TGC TTT TGG<\/span><\/p>\n

GB Env<\/span><\/p>\n

Oligo 1: <\/span>AA TTC AGA TCT ATG GAC TTT CTC ACC AAC CTC CGA TTC ACT GAG GGC CC<\/span><\/p>\n

Oligo 2: <\/span>GG GCC CTC AGT GAA TCG GAG GTT GGT GAG AAA GTC CAT AGA TCT G<\/span><\/p>\n","protected":false},"excerpt":{"rendered":"

Appendix Descriptions of plasmids used to express the Rel proteins studied. The source of the plasmid is indicated in parentheses. BC102:\u00a0 Vector alone (Kamens & Brent, 1991) BC102-Spe\/Sna:\u00a0 BC102 with linker containing SpeI and SnaBI sites at the unique EcoRI site (Dave & Gilmore, unpublished) JK v-Rel: Wild-type full-length v-Rel in BC102 (Kamens & Brent, […]<\/p>\n","protected":false},"author":4258,"featured_media":0,"parent":337,"menu_order":6,"comment_status":"closed","ping_status":"open","template":"","meta":[],"_links":{"self":[{"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/560"}],"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=560"}],"version-history":[{"count":5,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/560\/revisions"}],"predecessor-version":[{"id":612,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/560\/revisions\/612"}],"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=560"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}