Data Link 9

APPENDIX

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

Primers used for PCR amplification of REL exon 10 from tumors

REL 10-1 Forward: 5′-GTAAAATAAATTTTTCCTCCCACA-3′

REL 10-1 Reverse: 5′-CCATGCCATCAGCAGATTTA-3′

REL 10-2 Forward: 5′-GGTATCCCACCATTCCTGA-3′

REL 10-2 Reverse: 5′-GGATCTGACTTCAGTTGTGCAG-3′

REL 10-3 Forward: 5′-CAGTGACAGCATGGGAGAGA-3′

REL 10-3 Reverse: 5′TCTTTTAAATCTTGATACCACCT-3′

High Fidelity PCR was performed using 100 ng of genomic DNA from tissue in a final volume of 50 ul containing 200 nM dNTPs, 350 nM of each primer and 1.2 U of Expand High Fidelity PCR System in reaction buffer with 2 mM MgCl2.  PCRs were performed by an initial denaturation step at 95oC for 5 min, and then 30 cycles as follows:  95oC for 30 sec, 63oC to 57oC (-0.2oC per cycle) for 30 sec, 72oC for 40 sec; followed by a 10-min final elongation cycle at 72oC.  To promote heteroduplex formation prior to DHPLC analysis, the PCR products were heated at 95oC for 3 min, cooled to 65oC over 30 min, and were stored at 4oC.  Primers for amplification of REL exon 10 are shown directly above.  These oligonucleotides amplify three partially overlapping PCR products spanning the 770 base pair region of REL exon 10.  PCR products were then amplified on 2% agarose gels.

Primers used for PCR mutagenesis of REL

REL Forward:    5’-GAAGTTAGTGAATCTATCGATTTT-3’

REL-S525D Forward:  5’-CTGTTTTTGTTGATCAATCAGATG-3’

REL-S525D Reverse:  5’-CATCTGATTGATCAACAAAAACAG-3’

REL Forward (EcoRI, aa 500): 5′-CACGAATTCTGAGACAGCTCCATCAGATGTCCTCTTCC-3′

REL-Ser525 Reverse (HindIII): 5′-GCGAAGCTTTTACTCAAATGCATCTGATTGTGAAACAAA-3′

REL-Ala525 Reverse (HindIII): 5′-GCGAAGCTTTTACTCAAATGCATCTGATTGTGCAACAAAAAC-3′

Introduced mutations are underlined

Oligonucleotide used for kB probe (annealed and used in EMSA)

kB-WT: 5’-TCGAGAGGTCGGGAAATTCCCCCCCG-3’

kB site from chicken c-rel promoter is underlined

Retroviral vectors

JD214 BS+: Spleen necrosis virus vector (Sif et al., 1993)

JD-REL: JD214BS+ containing human REL subcloned as an XbaI-XhoI fragment into JD214BS+ digested with XbaI-SalI (Gilmore et al., 2001)

JD-RELD424-490: Spleen necrosis virus vector for expressing RELD424-490; JD214BS+ containing  human RELD424-490 subcloned as an XbaI-XhoI fragment into JD214BS+ digested with XbaI-SalI (Starczynowski et al., 2005)

JD-REL-S533,536A:  JD214BS+ containing REL-S533,536A; subcloned as an ApaI-NdeI fragment from pUC18-REL-S533,536A into JD-REL-D424-490 digested with ApaI-NdeI (Starczynowski et al., 2005)

JD-REL-S525P:    JD214BS+ containing REL-S525P; subcloned as an ApaI-NdeI fragment from pGEMT-REL-S525P into JD-RELD424-490 digested with ApaI-NdeI

JD-REL-S525D:  JD214BS+ containing REL-S525D; subcloned as an ApaI-NdeI PCR fragment into JD-RELD424-490 digested with ApaI-NdeI

Cloning Vectors

pGEM-T:  PCR subcloning vector (Promega, Madison, WI)

pGEMT-3’REL-S525P:  REL sequences encoding aa 300-587 were cloned from 2 human lymphomas into pGEM-T (Reiner Siebert, Kiel, Germany).

pBluescript SK+: Plasmid containing the MCS within lacZ (Stratagene).

pBluescript SK+-REL: Wild-type REL fragment (EcoRV-XhoI) subcloned into the corresponding sites (EcoRV-XhoI) of pBluescript SK+

pBluescript SK+-REL-S525D: S525D mutant REL fragment (EcoRV-XhoI) subcloned into the corresponding sites (EcoRV-XhoI) of pBluescript SK+

pBluescript SK+-RELD424-490: RELD424-490 EcoRV-XhoI fragment subcloned into EcoRV-XhoI sites of pBluescript SK+

pBluescriptSK+-3’REL-S525P: Plasmid containing REL fragment from EcoRV to 5’ end; REL-S525P fragment (EcoRV-HindIII) from JD-REL-S525P subcloned into the corresponding sites (EcoRV-HindIII) of pBluescript SK+

Expression Vectors

SG424: Expression plasmid containing GAL4 DNA-binding domain (DBD) upstream of MCS and downstream of SV40 promoter (Sadowski and Ptashne, 1989)

SG-3’REL: Wild-type human c-Rel (aa 278-587) fused to GAL4 DBD; pBluescript SK+-REL cut with BamHI-KpnI and was subcloned into BamHI-KpnI sites in SG424

SG-3’RELD424-490: REL (aa 278-423,491-587) fused to GAL4 DBD pBluescript SK+RELD424-490  cut with BamHI-KpnI and was subcloned into BamHI-KpnI sites in SG424

SG-3’REL-S533,536A: REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S533,536A cut with BamHI-KpnI and was subcloned into BamHI-KpnI sites in pSG424

SG-3’REL-S525P:  REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S525P cut with BamHI-KpnI and was subcloned into BamHI-KpnI sites in pSG424

SG-3’REL-S525D:  REL mutant fused to GAL4 DBD; pBluescript SK+-REL-S525D cut with BamHI-KpnI and was subcloned into BamHI-KpnI sites in pSG424

CM216: CMV promoter expression vector for full-length REL. Gift of Inder Verma, Salk Institute

pcDNA3.1(-): CMV promoter expression vector (Promega)

pcREL-S533,536A: pcDNA3.1(-) containing REL-S533,536A; REL-S533,536A was ubcloned as an XbaI-HindIII fragment from JD-REL-S533,536A into pcDNA3.1(-) digested with XbaI-HindIII

pcREL-S525P: pcDNA3.1(-) containing REL-S525P; REL-S525P was subcloned as an XbaI-HindIII fragment from JD-REL-S525P into pcDNA3.1(-) digested with XbaI-HindIII

pcRELD424-490:  pcDNA3.1(-) containing RELD424-490; RELD424-490 was subcloned as an XbaI-HindIII fragment from JD-RELD424-490 into pcDNA3.1(-) digested with XbaI-HindIII

pcDNA-FLAG-IKKalpha:  pcDNA containing FLAG-tagged human IKKalpha (Liang et al., 2003)

pcDNA-FLAG-IKKbeta:  pcDNA containing FLAG-tagged human IKKbeta (Liang et al., 2003)

pcDNA-GSK3beta:  pcDNA containing a constitutively active human GSK3beta.  Gift of Geoffrey Cooper (Boston University)

pGEX KG:  Expression plasmid containing GST domain upstream of MCS

pGEX-3’REL:   C-terminal aa of wild-type REL (aa 323-587) fused to GST; a REL PCR fragment was digested with EcoRI-HindIII and subcloned into pGEX-KG (EcoRI-HindIII) (Michael Garbati, Gilmore laboratory)

pGEX-3’REL-S525P:  REL-S525P (aa 323-587) fused to GST; a REL PCR fragment was digested with EcoRI-HindIII and was subcloned into pGEX-KG (EcoRI-HindIII) (Michael Garbati, Gilmore laboratory)

pGEX-3’REL: C-terminal aa of wild-type REL (aa 500-530) fused to GST; a REL PCR fragment was digested with EcoRI-HindIII and subcloned into pGEX-KG (EcoRI-HindIII)

pGEX-3’REL-S525A:  REL-S525A (aa 500-530) fused to GST; a REL PCR fragment was digested with EcoRI-HindIII and was subcloned into pGEX-KG (EcoRI-HindIII)

Reporter Plasmids

3x-kB-pGL2: 3x-kB-Luciferase-pGL2 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).

IkB-pGL2: IkBa-Luciferase-pGL2 reporter plasmid contains a 1.3 kb HindIII-EcoRI fragment of the chicken IkBa genomic clone, containing the transcriptional start site and 900 bp of upstream sequence placed upstream of the Luciferase  gene (Schatzle et al., 1995). Gift of Henry Bose (University of Texas, Austin)

SOD2-pGL3: The SOD2-Luciferase-pGL3 reporter plasmid contains 3.3 kb of the human SOD2 gene promoter and 0.4 kb of the SOD2 intronic enhancer placed upstream of the Luciferase gene (Abid et al, 2004).  Gift of R Abid (Harvard Medical School)

GAL4-site-Luc  Reporter plasmid containing 5 GAL4 sites upstream of minimal promoter and luciferase gene (Starczynowski et al., 2003). Gift of Joseph Lipsick (Stanford Medical School)

CMV-bgal:      CMV promoter–driven expression plasmid for b-galactosidase; used for normalization of transfection efficiency. (Starczynowski et al., 2003, 2005)

pGK-bgal:  SV40 promoter-driven expression plasmid for b-galactosidase; used for normalization of transfection efficiency  (Starczynowski et al., 2005)

Antisera

Western blotting

Rabbit anti-REL (C-terminal 15 aa; gift of Nancy Rice):  used at 1:10,000

Rabbit anti-MnSOD (Abcam Inc., Cambridge, MA): used at 1:2,000

Rabbit anti-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA): used at 1:500

Mouse anti-FLAG (Sigma, St Louis, MO):  used at 1:500

Indirect Immunofluorescence

Rabbit anti-REL primary (C-terminal 15 aa; gift of Nancy Rice):  1:50 dilution

FITC-conjugated goat anti-rabbit IgG secondary (Sigma):  1:80

EMSA Supershift

Rabbit (epitope near C terminus of RH domain; Kalaitzidis et al., 2002):  1 ul/reaction

References

Abid MR, IG Schoots, KC Spokes, SQ Wu, Mawhinney C and WC Aird (2004) Vascular endothelial growth factor-mediated induction of manganese superoxide dismutase occurs through redox-dependent regulation of forkhead and IkB/NF-kB. Journal of Biological Chemistry 279: 44030-44038

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

Kalaitzidis D, RE Davis, A Rosenwald, LM Staudt and TD Gilmore (2002) The human B-cell lymphoma cell line RC-K8 has multiple genetic alterations that dysregulate the Rel/NF-kB signal transduction pathway. Oncogene 21: 8759-8768

Liang M-C, S Bardhan, C Li, EA Pace, JA Porco Jr and TD Gilmore (2003) Jesterone dimer, a synthetic derivative of the fungal metabolite jesterone, blocks activation of transcription factor nuclear factor kB by inhibiting the inhibitor of kB kinase. Molecular Pharmacology 64: 123-131

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

Sadowski I and M Ptashne (1989) A vector for expressing GAL4(1-147) fusions in mammalian cells. Nucleic Acids Research 17: 7539

Schatzle JD, J Kralova and HR Bose Jr (1995) Avian IkBa is transcriptionally induced by c-Rel and v-Rel with different kinetics. Journal of Virology 69: 5383-5390

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

Starczynowski DT, JG Reynolds and TD Gilmore (2005) Mutations of tumor necrosis factor alpha-responsive serine residues within the C-terminal transactivation domain of human transcription factor REL enhance its in vitro transforming ability. Oncogene 24: 7355-7368