Data Link 12
Appendix of Plasmids and Primers for
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
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)
pGEM-REL-S484,494A: pGEM4 containing REL-S484,494A; an EcoRV-NdeI fragment from JD-REL-S484,494A was subcloned into pGEM-Hu-cRel digested with EcoRV-NdeI
Retroviral Vectors (used for subcloning)
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)
JD-REL-S484A: JD214 BS+ containing REL-S484A; an ApaI-HindIII fragment from pGEX-3’REL-S484A was subcloned into JD-REL digested with ApaI-HindIII
JD-REL-S484,494A: JD214 BS+ containing REL-S484,494A; an ApaI-HindIII fragment from pGEX-3’REL-S484,494A was subcloned into JD-REL digested with ApaI-HindIII
JD-REL-S484,494D: JD214 BS+ containing REL-S484,494D; an EcoRV-NdeI fragment from pSG-REL-S484,494D was subcloned into JD-REL digested with EcoRV-NdeI
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 (created by M. Garbati for Leeman et al, 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 (created by M. Garbati for Leeman et al, 2008)
pcDNA-FLAG-REL-S484,494A: pcDNA containing FLAG-tagged REL-S484,494A; an EcoRV-XhoI fragment from pGEM-REL-S484,494A was subcloned into pcDNA-FLAG-RHD-RID digested with EcoRV-XhoI
pcDNA-REL-S484,494D: pcDNA containing REL-S484,494D; an EcoRV-HindIII fragment from JD-REL-S484,494D was subcloned into pcDNA-REL digested with EcoRV-HindIII
pcDNA-FLAG-IKKa: pcDNA containing FLAG-tagged human IKKa (Starczynowski et al, 2007)
pcDNA-FLAG-IKKb: pcDNA containing FLAG-tagged human IKKb (Liang et al, 2003)
pcDNA-FLAG-IKKb-S177,181E: pcDNA containing FLAG-tagged human IKKb-S177,181E (Liang et al, 2006)
pcDNA-HA-IKKb-S177,181E: pcDNA containing HA-tagged human IKKb-S177,181E (gift of Sankar Ghosh, Yale University)
pcDNA-FLAG-IkBa-S32,36A: pcDNA containing FLAG-tagged human IkBa-S32,36A (gift of Susan Kandarian, Boston University)
pcDNA-mouse-RelA: pcDNA containing mouse RelA (Liang et al, 2006)
pGEX-based GST Fusion Protein Bacteria 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 (created by M. Garbati for Straczynowski et al, 2007)
pGEX-3’REL-S484A: REL-S484A (aa 323-587) fused to GST; overlapping PCR mutagenesis using pSG-REL-S484P as a template was used to mutate REL codon 484 to an Ala codon. The fragment containing REL-S484A was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII
pGEX-3’REL-S494A: REL-S494A (aa 323-587) fused to GST; overlapping PCR mutagenesis using pGEM-Hu-cRel as a template was used to mutate REL codon 494 to an Ala codon. The fragment containing REL-S494A was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII
pGEX-3’REL-S484,494A: REL-S484,494A (aa 323-587) fused to GST; overlapping PCR mutagenesis using pGEX-3’REL-S484A as a template was used to mutate REL codon 494 to an Ala codon. The fragment containing REL-S484,494A was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII
pGEX-3’RELD58: 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’RELD90: REL aa 323-497 fused to GST; a PCR fragment from pGEM-RELD90 (Starczynowski et al, 2003) was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII
pGEX-3’RELD110: REL aa 323-477 fused to GST; a PCR fragment from pGEM-RELD110 (Starczynowski et al, 2003) was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII
pGEX-3’RELD132: REL aa 323-455 fused to GST; a PCR fragment from pGEM-RELD132 (Starczynowski et al, 2003) was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII
pGEX-REL-aa-476-504: REL aa 476-504 fused to GST; a PCR fragment containing REL aa 476-504 was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII
pGEX-REL-aa-476-504-S484A: REL aa 476-504 S484A fused to GST; a PCR fragment containing REL aa 476-504 S484A was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII
pGEX-REL-aa-476-504-S494A: REL aa 476-504 S494A fused to GST; a PCR fragment containing REL aa 476-504 S494A was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII
pGEX-REL-aa-476-504-S484,494A: REL aa 476-504 S484,494A fused to GST; a PCR fragment containing REL aa 476-504 S484,494A was digested with EcoRI-HindIII and subcloned into pGEX-KG digested with EcoRI-HindIII
Expression Vectors for GAL4 Fusion Proteins in A293 Cells
pSG424: Expression vector with the SV40 early promoter controlling the GAL4 DNA-binding domain (aa 1-147) (DBD) upstream of the MCS (Sadowski & Ptashne, 1989)
pSG-REL: C-terminal sequences of wild-type REL (aa 278-587) fused to GAL4-DBD; a BamHI-KpnI fragment from pBluescript SK+-REL was subcloned into pSG424 digested with BamHI-KpnI (Starczynowski et al, 2003)
pSG-REL-S484P: C-terminal sequences of REL-S484P (aa 278-587) fused to GAL4-DBD; overlapping PCR mutagenesis using pGEM-Hu-cRel as a template was used to mutate REL codon 484 to a Pro codon. The fragment containing REL-S484P was digested with EcoRV-NdeI and subcloned into pSG-REL digested with EcoRV-NdeI (created by J. Leeman, Gilmore lab)
pSG-REL-S484A: C-terminal sequences of REL-S484A (aa 278-587) fused to GAL4-DBD; an EcoRV-NdeI fragment from JD-REL-S484A was subcloned into pSG-REL digested with EcoRV-NdeI
pSG-REL-S484,494A: C-terminal sequences of REL-S484,494A (aa 278-587) fused to GAL4-DBD; an EcoRV-NdeI fragment from JD-REL-S484,494A was subcloned into pSG-REL digested with EcoRV-NdeI
pSG-REL-S484D,S494A: C-terminal sequences of REL-S484D (aa 278-587) fused to GAL4-DBD; overlapping PCR mutagenesis using pSG-REL-S484,494A as a template was used to mutate REL codon 484 to an Asp codon. The fragment containing REL-S484D,S494A was digested with EcoRV-NdeI and subcloned into pSG-REL digested with EcoRV-NdeI
pSG-REL-S484,S494D: C-terminal sequences of REL-S484,494D (aa 278-587) fused to GAL4-DBD; overlapping PCR mutagenesis using pSG-REL-S484D,S494A as a template was used to mutate REL codon 494 to an Asp codon. The fragment containing REL-S484,494D was digested with EcoRV-NdeI and subcloned into pSG-REL digested with EcoRV-NdeI
pRG424: Expression vector with the RSV promoter controlling the GAL4-DBD upstream of the MCS; an NdeI/Klenow-HindIII fragment from pRSV-bgal was subcloned into pSG424 digested with PvuII-HindIII
pRG-REL: C-terminal sequences of wild-type REL (aa 278-587) fused to GAL4-DBD; an NdeI/Klenow-HindIII fragment from pRSV-bgal was subcloned into pSG-REL digested with PvuII-HindIII
pRG-REL-S484,494A: C-terminal sequences of REL-S484,494A (aa 278-587) fused to GAL4-DBD; an NdeI/Klenow-HindIII fragment from pRSV-bgal was subcloned into pSG-REL S484,494A digested with PvuII-HindIII
pcDNA-GAL4-REL: pcDNA containing REL aa 278-587 fused to the GAL4 DBD; a HindIII-XbaI fragment from pSG-REL was subcloned into pcDNA 3.1 (-) digested with HindIII-XbaI (created by J. Leeman, Gilmore lab)
Vertebrate Reporter Plasmids
pRSV-bgal: Contains the RSV LTR upstream of the b-galactosidase gene (gift of Douglas Faller, Boston University Medical School)
PolyA-GAL4E1b GAL4-luc: Contains GAL4 DNA-binding sites upstream and a minimal promoter upstream of the luciferase gene (gift of Joseph Lipsick, Stanford Medical School) (Starczynowski et al, 2003)
pGL3 Promoter Vector: a plasmid containing a partial SV40 promoter controlling the luciferase gene (Promega)
pSV40-Luc: Contains the luciferase gene controlled by the SV40 promoter sequences from pSG424; a PvuII-HindIII fragment from pSG424 was subcloned into pGL3 Promoter Vector digested with SmaI-HindIII
pRSV-Luc: Contains the luciferase gene controlled by the RSV promoter sequences from pRSV-bgal; an NdeI/Klenow-HindIII fragment from pRSV-bgal was subcloned into pGL3 Promoter Vector digested with SmaI-HindIII
Primers used for PCR
SP6 Promoter: 5’-GATTTAGGTGACACTATAG-3’
T7 Promoter: 5’-GTAATACGACTCACTATAGGGC-3’
REL-323-For-EcoRI: 5’-CCGGAATTCTAGGAGAAGGAAGATACTTC-3’
REL-FLAG-EcoRI-For: 5’-cgaattccatggcctccggtgc-3’
REL-423-Rev-BamHI: 5’-CGCAGGATCCCAATCATTCCCAACAGG-3’
REL-476-For-EcoRI: 5’-CACGAATTCGAGAGACTGATAATCCAAGACTTCTG-3’
REL-504-Rev-HindIII: 5’-GCGAAGCTTTTAGAGCTGTCTCAAGTCTCTTGGGTCTAAC-3’
REL-S484P-For: 5’-AATCCAAGACTTCTGCCCATGAATCTGA-3’
REL-S484P-Rev: 5’-GGTTTTCAAGATTCATGGGCAGAAGTCTT-3’
REL-P484A-For: 5’-AATCCAAGACTTCTGGCCATGAATCTTG-3’
REL-P484A-Rev: 5’-GGTTTTCAAGATTCATGGCCAGAAGTCTTG-3’
REL-S494A-For: 5’-AACCCCTCATGTAATGCAGTGTTAGACCC-3’
REL-S494A-Rev: 5’-CTCTTGGGTCTAACACTGCATTACATGAGG-3’
REL-A484D-For: 5’-AATCCAAGACTTCTGGACATGAATCTTG-3’
REL-A484D-Rev: 5’-GGTTTTCAAGATTCATGTCCAGAAGTCTTG-3’
REL-A494D-For: 5’-AACCCCTCATGTAATGACGTGTTAGACC-3’
REL-A494D-Rev: 5’-CTCTTGGGTCTAACACGTCATTACATGAGG-3’
Restriction sites and introduced mutations are underlined
Antisera
Western blotting
Rabbit anti-REL (C-terminal 15 aa; gift of Nancy Rice): used at 1:10,000 (see Kalaitzidis et al, 2002)
Immunoprecipitation
Mouse anti-FLAG (Sigma, St. Louis, MO; Catalog #A2220): conjugated to agarose beads
Mouse anti-NEMO (BD Pharmingen, San Jose, CA; Catalog #559675)
References
Barkett M, JE Dooher, L Lemonnier, L Simmons, JN Scarpati, Y Wang & 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
Gilmore TD, C Cormier, J Jean-Jacques & 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 & 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
Leeman JR, MA Weniger, TF Barth & TD Gilmore (2008) Deletion analysis and alternative splicing define a transactivation inhibitory domain in human oncoprotein REL. Oncogene, in revision
Liang M-C, S Bardhan, C Li, EA Pace, JA Porco Jr & 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
Liang M-C, S Bardhan, EA Pace, D Rosman, JA Beutler, JA Porco Jr & TD Gilmore (2006) Inhibition of transcription factor NF- B signaling proteins IKK and p65 through specific cysteine residues by epoxyquinone A monomer: correlation with its anticancer cell growth activity. Biochemical Pharmacology 71:634-645
Sadowski I & M Ptashne (1989) A vector for expressing GAL4(1-147) fusions in mammalian cells. Nucleic Acids Research 17: 7539
Sif S, AJ Capobianco & 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 & 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 & 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
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
