Data Link 13
Appendix of Plasmids and Primers for
Garbati MR, G Alço 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 T7 promoter elements (Promega)
pGEM-Hu-cRel: pGEM4 containing wild-type REL; an XbaI-XhoI/Klenow fragment of REL was subcloned into pGEM4 digested with XbaI-HincII (Barkett et al, 2001)
pBluescript-based Plasmids
pBluescript SK+: Plasmid containing the multiple cloning site (MCS) within lacZ (Stratagene)
pBS SK+ REL-RHD: pBluescript SK+ containing REL aa 305-323; an EcoRV-HindIII fragment from pGEX-5’REL was subcloned into pBluescript SK+ digested with EcoRV-HindIII
Retroviral Vectors
JD214 BS+: Spleen necrosis virus vector (Sif et al, 1993)
JD-REL: JD214 BS+ containing wild-type REL; an XbaI-XhoI REL fragment was subcloned into JD214 BS+ digested with XbaI-SalI (Gilmore et al, 2001)
pcDNA-based Expression Vectors
pcDNA 3.1(-): CMV promoter-driven expression vector (Invitrogen)
pcDNA-REL: pcDNA containing wild-type REL; an XbaI-HindIII REL fragment was subcloned into pcDNA 3.1(-) digested with XbaI-HindIII (Leeman et al., 2008)
pcDNA3-FLAG: pcDNA containing a FLAG tag upstream of the multiple cloning site (MCS)
pcDNA-FLAG-RHD-RID: 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)
pcDNA-FLAG-REL: 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)
pcDNA-FLAG-REL-RHD: 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
pcDNA-FLAG-RELDTAD1: pcDNA containing FLAG-tagged RELD424-490; 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)
pcDNA-FLAG-RELDTAD2: pcDNA containing FLAG-tagged RELD58; 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)
pcDNA-MYC-NEMO: pcDNA containing MYC-tagged NEMO (gift of Shigeki Miyamoto, University of Wisconsin, Madison, WI)
pcDNA-MYC-REL: 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)
pcDNA-MYC-REL-RHD: pcDNA containing MYC-tagged REL; an EcoRV-XbaI fragment from pSG-RELD282 (Starczynowski et al., 2003) was subcloned into MYC-REL digested with EcoRV and XbaI
pCMV-CBP: CMV-driven expression vector containing mouse CBP (gift of Ching-Chow Chen (National Taiwan University, Taipei, Taiwan)
pCMVb-p300-CHA: CMV-driven expression vector containing C-terminally HA-tagged human p300 (gift of Myles Brown, MIT)
pGEX-based GST Fusion Protein Bacterial Expression Vectors
pGEX KG: Expression plasmid containing GST domain upstream of MCS
pGEX-3’REL: 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
pGEX-5’REL: 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)
pGEX-3’RELDTAD1: RELD424-490 fused to GST; a PCR fragment from pGEM-RELD424-490 (Starczynowski et al., 2003) was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII
pGEX-3’RELDTAD2: 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
pGEX-3’RELDTAD1,2: REL aa 323-423 fused to GST; a PCR fragment from pGEM-RELD164 (Starczynowski et al., 2003) was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII
Vertebrate Reporter Plasmids
pRSV-bgal: Contains the RSV LTR upstream of the b-galactosidase gene (gift of Douglas Faller, Boston University Medical School)
3x-kB-Luciferase: pGL2-based reporter plasmid has a minimal c-fos 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)
Primers used for PCR for subcloning
SP6 Promoter: 5’-GATTTAGGTGACACTATAG-3’
T7 Promoter: 5’-GTAATACGACTCACTATAGGGC-3’
REL-323-For-EcoRI: 5’-CCGGAATTCTAGGAGAAGGAAGATACTTC-3’
REL-FLAG-EcoRI: 5’-cgaattccatggcctccggtgc-3’
REL-423-Rev-BamHI: 5’-CGCAGGATCCCAATCATTCCCAACAGG-3’
MYC-REL-For-EcoRI: 5’-CACGAATTCCGCCTCCGGTGCGTATAACC-3’
MYC-REL-Rev-XbaI: 5’-GCGTCTAGATTATACTTGAAAAAATTCATATGG-3’
Restriction sites are underlined.
Primers used for probe templates and quantitative RT-PCR
p300-5’probe-For: 5’-CACGAATTCCGGCCTAAACTCTCATCTCC-3’
p300-5’probe-Rev: 5’-CACAAGCTTCATTATCCCTTGTCCATTGC-3’
p300-exon3-4-For: 5’-GAGGAATGCCCAACATGG-3’
p300-exon11-12-Rev: 5’-TGGCCAAATTGATTCAAACC-3’
p300-exon31-For2: 5’-CACGAATTCGGTTGTGCAGCATACCAAGG-3’
p300-exon31-Rev2: 5’-CACGAATTCTGGCGTGAAGGATACTAAGC-3’
p300-qPCR-exon2-For: 5’-AACAGAGCAGTCCTGGATTAGG-3’
p300-qPCR-exon2-Rev: 5’-ATGGCAGGCTGATTTACTGG-3’
p300-qPCR-exon31-For: 5’-GCATATGCTCCCAAATCAGG-3’
p300-qPCR-exon31-Rev: 5’-AACTTGTCTGTGGGGAAACG-3’
GAPDH-qPCR-For: 5’-TGGTATCGTGGAAGGACTCATGAC-3’
GAPDH-qPCR-Rev: 5’-ATGCCAGTGAGCTTCCCGTTCAGC-3’
Primers used for quantitative PCR of genomic DNA
p300-GqPCR-exon13-For: 5’-CTCGTATGCAACAGCCTTCC-3’
p300-GqPCR-exon13-Rev: 5’-caagaatcaaaaataagtgaaaaatcc-3’
p300-GqPCR-exon14-For: 5’-ATTCACCCTCGCCTGTACC-3’
p300-GqPCR-exon14-Rev: 5’-GGTGTAGGTGTCTGCCTTGG-3’
p300-GqPCR-exon15-For: 5’-gcgtgtgtctcacctacttcc-3’
p300-GqPCR-exon15-Rev: 5’-CTGCTGGTTCTGGTTGATCC-3’
p300-GqPCR-exon16-For: 5’-TACCGAAACAGAAGAGAGAAGC-3’
p300-GqPCR-exon16-Rev: 5’-gctacctccagaacgaatgg-3’
p300-GqPCR-exon17-For: 5’-ACTACGACAGGCACTGATGC-3’
p300-GqPCR-exon17-Rev: 5’-GGGATTCCTAAAAGCTGAGG-3’
p300-GqPCR-exon18-For: 5’-GGCAGTATGTCGATGATATTTGG-3
p300-GqPCR-exon18-Rev: 5’-TGCCACAACAGTATCCAAGG-3’
p300-GqPCR-exon19-For: 5’-aatggtttcttttgcagTTGG-3’
p300-GqPCR-exon19-Rev: 5’-gcactccctggacatgtgg-3’
p300-GqPCR-exon20-For: 5’-gggctgtgttgtgtgaacg-3’
p300-GqPCR-exon20-Rev: 5’-aatgcagttacttacGTTTGAGG-3’
p300-GqPCR-exon21-For: 5’-gggtgaagtttgttcctttgg-3’
p300-GqPCR-exon21-Rev: 5’-ggatcatctgattggtcatgc-3’
p300-GqPCR-exon22-For: 5’-gctgtctttgtcagaagtcatgg-3’
p300-GqPCR-exon22-Rev: 5’-CAGATGATCTCATGGTGAAGG-3’
p300-GqPCR-exon23-For: 5’-caacggtttatctaagttgtgtaagc-3’
p300-GqPCR-exon23-Rev: 5’-tttggatggtttcttatctaggc-3’
p300-GqPCR-exon24-For: 5’-cctagGGTTGCCATCTACCA-3’
p300-GqPCR-exon24-Rev: 5’-tttggatccacgaggagaag-3’
p300-GqPCR-exon25-For: 5’-TGTGGACAGTGGAGAGATGG-3’
p300-GqPCR-exon25-Rev: 5’-GAGGGCAGTCAGAGCCATAC-3
p300-GqPCR-exon27-For: 5’-TTACACAACAGGGCATATTTGG-3’
p300-GqPCR-exon27-Rev: 5’-CTTGTAGTCATGGACAATACGC-3
p300-qPCR-exon31-For: 5’-GCATATGCTCCCAAATCAGG-3’
p300-qPCR-exon31-Rev: 5’-AACTTGTCTGTGGGGAAACG-3’
Antisera
Western blotting
rabbit anti-REL (C-terminal 15 aa, #265, Nancy Rice, NCI, Frederick, MD) used at 1:10,000
rabbit anti-REL (NLS, #1206, Nancy Rice, NCI) used at 1:2500
rabbit anti-CBP (#4772, Cell Signaling Technology, Danvers, MA) used at 1:1000
mouse anti-CBP (sc-7300, Santa Cruz Biotechnology, Santa Cruz, CA) used at 1:200
rabbit anti-p300 (N-terminal, sc-584, Santa Cruz Biotechnology) used at 1:200
rabbit anti-p300 (C-terminal, sc585, Santa Cruz Biotechnology) used at 1:200
rabbit anti-IkBa (sc-371, Santa Cruz Biotechnology) used at 1:500
rabbit b-tubulin (sc-9104, Santa Cruz Biotechnology) used at 1:500
rabbit anti-HA (sc-805, Santa Cruz Biotechnology) used at 1:500
rabbit anti-FLAG (#2368, Cell Signaling Technology) used at 1:1000
mouse anti-MYC (sc-40, Santa Cruz Biotechnology) used at 1:200
Immunoprecipitation
mouse anti-FLAG (#A2220, Sigma, St. Louis, MO) conjugated to agarose beads; used 40 ml/ml
rabbit anti-p300 (N-terminal, sc-584, Santa Cruz Biotechnology) used 6 mg/ml
rabbit normal IgG (sc-2027, Santa Cruz Biotechnology) used 6 mg/ml
mouse anti-MYC (sc-40, Santa Cruz Biotechnology) used 6 mg/ml
References
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
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
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
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
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
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
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
