{"id":576,"date":"2011-04-26T15:22:51","date_gmt":"2011-04-26T19:22:51","guid":{"rendered":"https:\/\/www.bu.edu\/nf-kb\/?page_id=576"},"modified":"2011-04-28T18:32:52","modified_gmt":"2011-04-28T22:32:52","slug":"data-link-10","status":"publish","type":"page","link":"https:\/\/www.bu.edu\/nf-kb\/the-gilmore-lab\/data-link-10\/","title":{"rendered":"Data Link 10"},"content":{"rendered":"

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

Plasmids and oligonucleotides used in<\/p>\n

Herscovitch M, W Comb, T Ennis, K Coleman, S Yong, B Armstead, D Kalaitzidis, S Chandani & TD Gilmore. 2007. Intermolecular disulfide bond formation in the NEMO dimer requires Cys54 and Cys347. Biochemical and Biophysical Research Communications 367: 103-108<\/p>\n

Intermediate Subcloning Plasmids <\/strong><\/p>\n

pGEM4\u00a0\u00a0 (Promega)<\/p>\n

pGEM4-NEMO:\u00a0 pcDNA-6\u00d7Myc-NEMO was digested with EcoRI\/XbaI and the fragment was subcloned into the corresponding sites of pGEM4<\/p>\n

pGEM4-NEMO-C54\/347A:\u00a0 pcDNA-6\u00d7Myc-NEMO-C54\/347A was digested with EcoRI\/XbaI and the fragment was subcloned into the corresponding sites of pGEM4<\/p>\n

Retroviral Vectors <\/strong><\/p>\n

pBABE:\u00a0 Retroviral vector containing the puromycin resistance cassette (Morgenstern and Land, 1990)<\/p>\n

pBABE-NEMO: pGEM4-NEMO was digested with EcoRI\/SalI and the digested fragment was subcloned into the corresponding sites of pBABE<\/p>\n

pBABE-NEMO-C54\/347A: pGEM4-NEMO-C54\/347A was digested with EcoRI\/SalI and thedigested fragment was subcloned into the corresponding sites of pBABE<\/p>\n

Expression Vectors <\/strong><\/p>\n

pcDNA-6\u00d7Myc-NEMO: Gift of S.Miyamoto (University of Wisconsin, Madison)<\/p>\n

pcDNA-6\u00d7Myc-NEMO-C54A: Site-directed mutagenesis was performed using two synthetic complementary oligonucleotide primers containing the relevant point mutation and two flanking primers (5\u2019 and 3\u2019). \u00a0The final PCR product was digested with HindIII and EcoRV and used to replace the corresponding HindIII\/EcoRV fragment in pcDNA-6\u00d7Myc-NEMO<\/p>\n

pcDNA-6\u00d7Myc-NEMO-C347A: Site-directed mutagenesis was performed as described above. The final PCR product was digested with EcoRV\/XbaI and used to replace the corresponding EcoRV\/XbaI fragment in pcDNA-6\u00d7Myc-NEMO<\/p>\n

pcDNA-6\u00d7Myc-NEMO-C54\/347A:\u00a0 pcDNA-6\u00d7Myc-NEMO-C347A was digested with EcoRV\/XbaI and this fragment was used to replace the corresponding EcoRV\/XbaI fragment in the pcDNA-6\u00d7Myc-NEMO-C54A plasmid<\/p>\n

pcDNA3-FLAG: Gift of Bakary Sylla (Lyons, France)<\/p>\n

pcDNA3-FLAG-NEMO:\u00a0 Gift of Sankar Ghosh (Yale University)<\/p>\n

pcDNA3-FLAG-NEMO-C54A:\u00a0 PCR amplification of pcDNA-6\u00d7Myc-NEMO-C54A\u00a0 was performed using oligonucleotides with inserted EcoRI and BamHI sites and the PCR product was further digested with EcoRI\/BamHI. The digested fragment was then subcloned into the corresponding sites of pcDNA-FLAG.<\/p>\n

pcDNA3-FLAG-NEMO-C347A:\u00a0 PCR amplification of pcDNA-6\u00d7Myc-NEMO-C347A\u00a0 was performed using oligonucleotides with inserted EcoRI and BamHI sites and the PCR product was further digested with EcoRI\/BamHI. The digested fragment was then subcloned into the corresponding sites of pcDNA-FLAG.<\/p>\n

pcDNA3-FLAG-NEMO-C54\/347A: PCR amplification of pcDNA-6\u00d7Myc-NEMO-C54\/347A\u00a0 was performed using oligonucleotides with inserted EcoRI and BamHI sites and the PCR product was further digested with EcoRI\/BamHI. The digested fragment was then subcloned into the corresponding sites of pcDNA-FLAG.<\/p>\n

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

pcDNA-3\u00d7kB-luciferase: kB site-luciferase reporter plasmid (Starczynowski et al., 2007)<\/p>\n

pRSV-bgal: Expression of the b-galactosidase gene driven by the Rous sarcoma virus promoter. Gift of Dr Douglas Faller (Boston University Medical School)<\/p>\n

Primers used for amplification of NEMO from pcDNA-6\u00d7Myc <\/strong><\/p>\n\n\n\n\n
FLAG-NEMO-EcoRI-FOR <\/strong><\/td>\nGATAGAATTC<\/strong><\/span>AAATAGGCACCTCTGGAAGAGC<\/td>\n<\/tr>\n
FLAG-NEMO-BamHI-REV <\/strong><\/td>\nAATGGATCC<\/strong><\/span>CTACTCAATGCACTCCATGACATG<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

*Restriction sites in bold<\/p>\n

Primers used for site-directed mutagenesis of pcDNA-6\u00d7Myc-NEMO <\/strong><\/p>\n\n\n\n\n\n\n
NEMO-C54A-FOR <\/strong><\/td>\nGCGCGCC<\/strong>CTGGAGGAGAATCAAG<\/td>\n<\/tr>\n
NEMO-C54A-REV <\/strong><\/td>\nTGATTCTCCTCCAGGGC<\/strong>GCGC<\/td>\n<\/tr>\n
NEMO-C347A-FOR <\/strong><\/td>\nAAGGCC<\/strong>AGCGCTCAGGAG<\/td>\n<\/tr>\n
NEMO-C347A-REV <\/strong><\/td>\nCTCCTGAGCGCTGGC<\/strong>CTTCC<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Mutant codons in bold<\/p>\n

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

Morgenstern JP, Land H. (1990). Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell. Nucleic Acids Research 18: 3587-3596<\/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 Plasmids and oligonucleotides used in Herscovitch M, W Comb, T Ennis, K Coleman, S Yong, B Armstead, D Kalaitzidis, S Chandani & TD Gilmore. 2007. Intermolecular disulfide bond formation in the NEMO dimer requires Cys54 and Cys347. Biochemical and Biophysical Research Communications 367: 103-108 Intermediate Subcloning Plasmids pGEM4\u00a0\u00a0 (Promega) pGEM4-NEMO:\u00a0 pcDNA-6\u00d7Myc-NEMO was digested with […]<\/p>\n","protected":false},"author":4258,"featured_media":0,"parent":337,"menu_order":14,"comment_status":"closed","ping_status":"open","template":"","meta":[],"_links":{"self":[{"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/576"}],"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=576"}],"version-history":[{"count":3,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/576\/revisions"}],"predecessor-version":[{"id":602,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/576\/revisions\/602"}],"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=576"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}