Data Link 14

Appendix of Plasmids, Primers and Antibodies for

Garbati MR, Gilmore TD (2011) A rearranged EP300 gene in the human B-cell lymphoma cell line RC-K8 encodes a disabled transcriptional co-activator that contributes to cell growth and oncogenicity. Cancer Letters 302: 76-83

pGEX-based GST Fusion Protein Bacterial Expression Vectors

pGEX KG: Expression plasmid containing GST domain upstream of multi-cloning site (MCS)

pGEX-3’REL: GST fused to the C-terminal transactivation domain sequences of wild-type REL (aa 324-587); a PCR fragment using pGEM-Hu-cRel as a template was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII (Garbati and Gilmore, 2008)

pGEX-VP16: GST fused to the C-terminal transactivation domain sequences of VP16 (aa 452-490) (Uesugi et al., 1997; gift of Gregory Verdine, Harvard University)

pcDNA-based Expression Vectors

pcDNA 3.1(-): CMV promoter-driven expression vector (Invitrogen)

pcDNA-REL: pcDNA containing a full-length REL cDNA; an XbaI-HindIII REL fragment was subcloned into pcDNA 3.1(-) that was digested with XbaI-HindIII (Leeman et al., 2008)

pCMVβ-p300: CMV-driven expression vector containing C-terminally HA-tagged human p300 (gift of Myles Brown, MIT) (Garbati et al., 2010)

pCMVβ-p300ΔC: CMV-driven expression vector containing p300ΔC from RC-K8 cells; a PCR fragment generated using p300-For-ex9 and non-p300-gene-Hind3-Rev as primers and RC-K8 cell total cDNA as a template was digested with NdeI and HindIII and was subcloned in a three-way ligation with a (5’) NotI-NdeI fragment from pCMVβ-p300 into pCMVβ-p300 digested with NotI and HindIII

Vertebrate Reporter Plasmids

pRSV-βgal: Contains the RSV LTR upstream of the β-galactosidase gene (gift of Douglas Faller, Boston University Medical School)

3x-κB-Luciferase: pGL2-based reporter plasmid has a minimal c-fos promoter element and three copies of the major histocompatibility complex (MHC) class κB-site element (TGGGGATTCCCCA) placed upstream of the luciferase gene (Mitchell and Sugden, 1995)

Retroviral Vectors

pSIREN-RetroQ: CMV/MSV-driven retroviral vector designed to express shRNA (Clontech, Mountain View, CA)

pSIREN-p300: pSIREN containing shRNA sequences directed against 5’ sequences of EP300 (5′-ACCAGATGCCTCGAATAA-3′; Sankar et al., 2008); inserted sequences were designed using the shRNA Sequence Designer (Clontech) with BamHI and EcoRI overhangs and subcloned into pSIREN-RetroQ digested with BamHI and EcoRI.

pSIREN-control: pSIREN containing non-targeting shRNA sequences (5’-GCAAGCTGCCCGTGCCCTG-3’; Scherr et al., 2003); inserted sequences were designed using the shRNA Sequence Designer (Clontech) with BamHI and EcoRI overhangs and subcloned into pSIREN-RetroQ digested with BamHI and EcoRI.

pCL-10a1: Retrovirus packaging vector (Imgenex, San Diego, CA)

Oligonucleotides for Creating shRNA Retroviral Vector Plasmids





Targeting sequences are italicized. Hairpin sequences are underlined.

Primers for 3’RACE of the p300ΔC cDNA from RC-K8 Cell Total cDNA




(N = A, C, G, or T; V = A, G, or C)

Universal Primer A Mix (Clontech):



Nested Universal Primer A (Clontech):  5’-AAGCAGTGGTATCAACGCAGAGT-3’

Primers for PCR Amplification for Subcloning of the p300ΔC 3’ End into pCMVβ-p300



Restriction sites are underlined.

Primers for RT-PCR and PCR of Genomic DNA of p300ΔC for DNA Sequencing

p300-For-ex17:  5’-ACTACGACAGGCACTGATGC-3’

non-p300-gene-Rev:  5’-CATCTGCTCAATGACCACAG-3’


Western Blotting

rabbit anti-p300 (N-terminal, sc-584, Santa Cruz Biotechnology, Santa Cruz, CA) used at 1:200

rabbit anti-β-tubulin (sc-9104, Santa Cruz Biotechnology) used at 1:500

Indirect Immunofluoresence

rabbit anti-p300 (N-terminal, sc-584, Santa Cruz Biotechnology) used at 1:40

rabbit IgG (whole molecule) FITC-conjugated (F9887, Sigma, St. Louis, MO) used at 1:80


Garbati MR, Gilmore TD (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

Garbati M, Alço G, Gilmore TD. 2010. The histone acetyltransferase p300 co-activator is functionally inactivated in the human diffuse large B-cell lymphoma cell line RC-K8. Cancer Letters 291:237-245

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

Mitchell T, Sugden B (1995) Stimulation of NF-κB-mediated transcription by mutant derivatives of the latent membrane protein of Epstein-Barr virus. Journal of Virology 65:2968-2976

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold  Spring Harbor, NY, Cold Spring Harbor Press

Sankar N, Baluchamy S, Kadeppagari R-K, Singhal G, Weitzman S (2008) p300 provides a corepressor function by cooperating with YY1 and HDAC3 to repress c-Myc. Oncogene 27:5717-5728

Scherr M, Battmer K, Ganser A, Eder M (2003) Modulation of gene expression by lentiviral-mediated delivery of small interfering RNA. Cell Cycle 2:251-257

Uesugi M, Nyanguile O, Lu H, Levine AJ, Verdine GL (1997) Induced alpha helix in the VP16 activation domain upon binding to a human TAF. Science 277:1310-1313