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

p300-shRNA-Sense:  5’-GATCCACCAGATGCCTCGAATAATTCAAGAGATTATTCGAGGCATCATCTGGTTTTTTTACGCGTG-3’

p300-shRNA-Antisense:  5’-AATTCACGCGTAAAAAAACCAGATGATGCCTCGAATAATCTCTTGAATTATTCGAGGCATCATCTGGTG-3’

Control-shRNA-Sense:  5’-GATCCGCAAGCTGCCCGTGCCCTGTTCAAGAGACAGGGCACGGGCAGCTTGCTTTTTTACGCGTG-3’

Control-shRNA-Antisense:  5’-AATTCACGCGTAAAAAAGCAAGCTGCCCGTGCCCTGTCTCTTGAACAGGGCACGGGCAGCTTGCG-3’

Targeting sequences are italicized. Hairpin sequences are underlined.

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

p300-RACE-ex15:  5’-GCCATTGCTGAGAAGCAGCCTTCC-3’

p300-RACE-ex16:  5’-CCCAGTCATCTCCGGCTCCAGGAC-3’

3’RACE CDS Primer A (Clontech):  5’-AAGCAGTGGTATCAACGCAGAGTAC(T)₃₀VN-3’

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

Universal Primer A Mix (Clontech):

UPM Long:  5’-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3’

UPM Short:  5’-CTAATACGACTCACTATAGGGC-3’

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

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

p300-For-ex9:  5’-GCTTTAAAAGACAGACGGATGG-‘3

non-p300-gene-Hind3-Rev:  5’-CCCAAGCTTCAAAGCCACAGAAATTGCATCTC-3’

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’

Antisera

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

References

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