{"id":564,"date":"2011-04-26T15:12:30","date_gmt":"2011-04-26T19:12:30","guid":{"rendered":"https:\/\/www.bu.edu\/nf-kb\/?page_id=564"},"modified":"2011-04-26T15:12:30","modified_gmt":"2011-04-26T19:12:30","slug":"data-link-1","status":"publish","type":"page","link":"https:\/\/www.bu.edu\/nf-kb\/the-gilmore-lab\/data-link-1\/","title":{"rendered":"Data Link 1"},"content":{"rendered":"

APPENDIX <\/strong><\/p>\n

Epinat J-C, Dvorin EL and Gilmore TD. (2000) Envelope-dependent transactivation by the retroviral oncoprotein is required for transformation of chicken spleen cells. Oncogene 19 :3131-3137<\/p>\n

A.\u00a0 Descriptions of plasmids used in this paper. <\/strong> (The source of the plasmid is indicated in parentheses.)<\/p>\n

1.\u00a0 Plasmids for the expression of Rel proteins in yeast.<\/strong><\/p>\n

BC102:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Vector alone with Adh promoter, Trp-selectable (Kamens & Brent, 1991)<\/p>\n

BC102-Spe\/Sna:\u00a0 BC102 with linker containing SpeI and SnaBI sites at the unique EcoRI site
\n(Dave & Gilmore, unpublished; see Epinat et al., 2000a)<\/p>\n

BC 5\u2019v-Rel:\u00a0\u00a0\u00a0 Contains sequences up to v-rel\u2019s unique Hinc II restriction site, includes<\/p>\n

mainly the N-terminal Rel Homology domain and lacking the C-<\/p>\n

terminal transactivation domains; from amino acids (aa) 1-331 (Mosialos &<\/p>\n

Gilmore, 1993; see Epinat et al., 2000a)<\/p>\n

BC5′-RevA-vRel:\u00a0 Identical to BC-5’v-Rel, but contains the Envelope sequences of the Rev-A helper virus Env (this study)<\/p>\n

BC5’v-N6D:\u00a0 Identical to BC-5’v-Rel but contains an Asn to Asp mutation at amino acid 6 of the Env (this study)<\/p>\n

BC5’3,9L-RevA-vRel:\u00a0 Identical to BC-RevA-v-Rel but has Cys->Leu and Ser->Leu mutations at aa 3 and 9, respectively (this study)<\/p>\n

BC5’3,9Y-RevA-Rel:\u00a0 Identical to BC-RevA-v-Rel but has Cys->Tyr and Ser->Tyr mutations at aa 3 and 9, respectively (this study)<\/p>\n

BC 5’DEnv-v-Rel :\u00a0 A 5’v-Rel protein that has a deletion of aa 1-11 (Envelope sequence) and the addition of an AUG start codon (Epinat et al., 2000a)<\/p>\n

IgK6:\u00a0 LacZ reporter plasmid with 6 upstream kB sites, Ura-selectable (Epinat et al., 2000a)<\/p>\n

2.\u00a0 Plasmids used to express the GAL4-Rel proteins in yeast.<\/strong><\/p>\n

GBT9: GAL4 DNA-binding domain alone, aa 1-147 (Clontech), Trp-selectable<\/p>\n

GB 5\u2019v-Rel: 5\u2019v-Rel from aa 2-331 fused to GAL4 DNA-binding domain (Epinat et al., 2000a)<\/p>\n

GB-5’RevA-vRel:\u00a0 identical to GB5’v-Rel except has aa 1-331 of v-Rel fused to the GAL4-DNA binding domain, and has the Rev-A Env aa substitutions at aa 3, 6, and 9<\/p>\n

GB-F3C-vRel, GB-F9S-vRel, GB-F3C\/F9S-vRel, GB-N6D-v-Rel, GB-F3C\/N6D-vRel, GB-N6D\/F9S-vRel, GB-3,9L-RevA-vRel, GB-3,9Y-RevA-vRel, GB-C3L\/S9F-RevA-vRel, GB-C3F\/S9L-RevA-vRel, GB-C3Y\/S9F-RevA-vRel, GB-C3F\/S9Y-RevA-v-Rel, GB-F10-v-Rel, GB-S10-vRel:\u00a0 All are identical to GB-5’RevA-vRel except with the substitutions as described in Figures 2a and 3a of the manuscript<\/p>\n

GB 5\u2019-Ch-cRel: 5\u2019c-Rel from aa 1-322 fused to GAL4 DNA-binding domain (Epinat et al., 2000a)<\/p>\n

GB-5′-Env-Ch-cRel: Chicken c-Rel from aa 2-322 with 11 N-terminal Env aa of v-Rel and fused to GAL4 DNA-binding domain (Epinat et al., 2000a)<\/p>\n

GB-5′-Hu-cRel:\u00a0 Human c-Rel from aa 1-322 fused to GAL4 DNA-binding domain (this study)<\/p>\n

GB5′-Env-Hu-cRel:\u00a0 Human c-Rel from aa 2-322 with 11 N-terminal Env aa of v-Rel and fused to GAL4 DNA-binding domain (this study)<\/p>\n

GB 5’DEnv-v-Rel : A GAL4-5’v-Rel protein which consists of a deletion of aa 1-11, v-Rel Envelope sequence (Epinat et al., 2000a)<\/p>\n

SD5-Leu:\u00a0 GAL4 site lacZ reporter plasmid, Leu-selectable (Epinat et al., 2000a)<\/p>\n

3.\u00a0 Plasmids used to express aa 1-331 of v-Rel mutants in vitro.<\/strong><\/p>\n

Plasmids for the expression of aa 1-331 of wild-type v-Rel and RevA-vRel are described in Epinat et al. (2000a).\u00a0 These plasmids have a stop codon after aa 331 of v-Rel.<\/p>\n

Plasmids for the expression of aa 1-331 of N6D-vRel, 3,9L-RevA-vRel, 3,9Y-RevA-vRel, and DEnv-vRel are identical to the pGEM-v-Rel and pGEM-RevA-vRel plasmids, except with mutations in the N-terminal Env sequences.<\/p>\n

4.\u00a0 Plasmids used to express the GAL4-Rel proteins in CEF.<\/strong><\/p>\n

SG424: SV40 promoter plasmid controlling expression of GAL4 (1-147) (see Epinat et al., 2000a).<\/p>\n

SG-5-vRel:\u00a0 GAL4 fused to a portion of v-Rel encoding aa 2-331 (see Epinat et al., 2000a).<\/p>\n

All other SG-mutant Env-v-Rel plasmids are as described above for the GBT9-based plasmids, and they were constructed by subcloning XhoI-BamHI fragments from the GBT9-based plasmids into SG-5’RevA-Rel (from Epinat et al., 2000a).<\/p>\n

5.\u00a0 Plasmids used to express full-length v-Rel mutants in spleen cell transformation assays.<\/strong><\/p>\n

JD-v-Rel (GM282BS+):\u00a0 Spleen necrosis virus vector for the expression of wild-type v-rel (Sif et al., 1993)<\/p>\n

JD-RevA-v-Rel:\u00a0 Spleen necrosis virus vector for the expression of RevA-vRel (v-Rel with Rev-A Env aa at its N terminus) (this study)<\/p>\n

JD-vN6D, JD-3,9L-RevA-v-Rel, JD-3,9Y-RevA-vRel, JD-3,9P-RevA-v-Rel:\u00a0 Spleen necrosis virus vectors for the expression of the indicated v-Rel mutants (this study)<\/p>\n

B. Construction of plasmids used in this paper.<\/strong><\/p>\n

1.\u00a0 Plasmids with citations in parenthesis above were constructed previously.<\/p>\n

2.\u00a0 Mutant Rel plasmids used in this study were all constructed in the following order:<\/p>\n

1) PCR mutagenesis was used to create the desired Env mutants.\u00a0 For the Env mutants, PCR was performed with an upstream mutagenic primer (see below) and a downstream Rev-Rel primer (5′-CTA TAC GTA CTA GTT GAC AGT GGG GAT TGG AGC TGC TTT TGG-3′; see also below) on GB 5\u2019v-Rel: 5\u2019v-Rel or GB-5’RevA-vRel;<\/p>\n

2) The PCR product was digested with XmaI and ClaI and the 450 bp fragment was used to replace the corresponding fragment of GB-5’v-Rel (which was also digested with XmaI and ClaI);<\/p>\n

3)\u00a0 The GB-derived vector was sequenced with a GAL4-annealing primer (5′-ACAGCATAGAATAAGTG-3′) to confirm the identity of the mutation, to confirm the precision of the GAL4-Rel fusion point, and to screen for other spurious mutations; and<\/p>\n

4)\u00a0\u00a0 The correct GB-mutant Rel plasmid was then digested with XmaI-StuI and these fragments were used to replace the corresponding fragments in a pGEM5’v-Rel fragment or in JD-v-Rel.\u00a0 Correct clones were identified by size and by digestion with XbaI (since a correct subcloning would remove a 5′ XbaI site in these clones).\u00a0 In addition, the GBT9-based subclone was digested with XhoI-BamHI and the fragment was used to replace the corresponding fragment in SG-5’RevA-vRel (described in Epinat et al., 2000a).\u00a0 Finally, for expression of aa 1-331 of certain Env Rel mutant proteins (5’Rev-Rel; 5’v-N6D; 5’3,9L-RevA-vRel; 5’3,9Y-RevA-v-Rel; 5’DEnv-vRel) in yeast, the corresponding GB-mutant Rel plasmids were digested with SmaI and HincII and the fragment was subcloned into the SnaBI site of BC102-Spe\/SnaBI.<\/p>\n

3.\u00a0 Primers used for Env mutagenesis of v-Rel:<\/strong><\/p>\n

a.\u00a0 Upstream Env primers for mutagenesis:<\/p>\n

RevA2 (RevA-vRel)
\nXmaI\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Cys\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Asp\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Ser
\nCCCCCCGGGG ATG GAC TGT CTC ACC GAC CTC CGA TCC ACT GAG<\/p>\n

RevA3 (F3C-vRel)
\nCys\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Asn\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Phe
\nCCCCCCGGGG ATG GAC TGT CTC ACC AAC CTC CGA TTC ACT GAG<\/p>\n

RevA4 (N6D-vRel)
\nPhe\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Asp\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Phe
\nCCCCCCGGGG ATG GAC TTT CTC ACC GAC CTC CGA TTC ACT GAG<\/p>\n

RevA5 (F9S-vRel)
\nPhe\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Asn\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Ser
\nCCCCCCGGGG ATG GAC TTT CTC ACC AAC CTC CGA TCC ACT GAG<\/p>\n

RevA6 (S9F-RevA-vRel)
\nCys\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Asp\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Phe
\nCCCCCCGGGG ATG GAC TGT CTC ACC GAC CTC CGA TTC ACT GAG<\/p>\n

RevA7 (D6N-RevA-vRel)
\nCys\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Asn\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Ser
\nCCCCCCGGGG ATG GAC TGT CTC ACC AAC CTC CGA TCC ACT GAG<\/p>\n

RevA8 (C3F-RevA-vRel)
\nPhe\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Asp\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Ser
\nCCCCCCGGGG ATG GAC TTT CTC ACC GAC CTC CGA TCC ACT GAG<\/p>\n

RevA9 (3,9L-RevA-vRel)
\nLeu\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Asp\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Leu
\nCCCCCCGGGG ATG GAC TTG CTC ACC GAC CTC CGA TTG ACT GAG<\/p>\n

RevA10 (3,9Y-RevA-vRel)
\nTyr\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Asp\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Tyr
\nCCCCCCGGGG ATG GAC TAT CTC ACC GAC CTC CGA TAC ACT GAG<\/p>\n

RevA11 (F10-vRel)
\nMet———————-10 Phe———————————-\u00a0 Gly\u00a0\u00a0\u00a0 Ile
\nCCCCCCGGGG ATG TTC TTT TTC TTC TTC TTC TTC TTC TTT TTC GGT ATC TC<\/p>\n

RevA12 (S10-vRel)
\nMet———————-10 Ser—————————————\u00a0 Gly\u00a0\u00a0 Ile
\nCCCCCCGGGG ATG TCC AGT AGC AGC TCC TCC AGT TCC TCT TCG GGT ATC TC<\/p>\n

RevA14 (C3F\/S9L-RevA-vRel)
\nPhe\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Asp\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Leu
\nCCCCCCGGGG ATG GAC TTT CTC ACC GAC CTC CGA TTG ACT GAG<\/p>\n

RevA15 (C3Y\/S9F-RevA-vRel)
\nTyr\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Asp\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Phe
\nCCCCCCGGGG ATG GAC TAT CTC ACC GAC CTC CGA TTC ACT GAG<\/p>\n

RevA16 (C3F\/S9Y-RevA-vRel)
\nPhe\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Asp\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Tyr
\nCCCCCCGGGG ATG GAC TTT CTC ACC GAC CTC CGA TAC ACT GAG<\/p>\n

RevA26 (3,9P-RevA-vRel)
\nPro\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Asp\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Pro
\nCCCCCCGGGG ATG GAC CCT CTC ACC GAC CTC CGA CCC ACT GAG<\/p>\n

b.\u00a0 Downstream primter<\/p>\n

Rev-Rel (reverse oligo, around the Hinc2 site of v-Rel, used for all constructs above):<\/p>\n

|\u00a0 SnaB1\u00a0 |\u00a0 STOP |\u00a0\u00a0 Hinc2\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 ——————-vRel———————|
\nCTA TAC GTA CTA GTT GAC AGT GGG GAT TGG AGC TGC TTT TGG<\/p>\n

4.\u00a0 Primers used for PCR amplification and mutagenesis of human c-Rel:<\/strong><\/p>\n

a.\u00a0 Upstream primer for Env-Hu-c-Rel (c-Rel starts at 3rd codon):<\/p>\n

Xma1\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Met\u00a0\u00a0 Asp\u00a0 Phe\u00a0\u00a0 Leu\u00a0 Thr\u00a0\u00a0\u00a0 Asn\u00a0\u00a0 Leu\u00a0 Arg\u00a0 Phe\u00a0\u00a0 Thr\u00a0 Glu\u00a0\u00a0 —human c-Rel—————-
\n5’CCCCCCGGGG ATG GAC TTT CTG ACG AAC CTA CGC TTC ACC GAG ggt gcg tat aac ccg tat ata gag3′<\/p>\n

b.\u00a0 Upstream primer for Rev-hu-c-Rel (c-Rel starts at 3rd codon)<\/p>\n

Xma1\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Met\u00a0\u00a0 Asp\u00a0 Cys\u00a0\u00a0 Leu\u00a0 Thr\u00a0\u00a0\u00a0 Asp\u00a0\u00a0 Leu\u00a0 Arg\u00a0 Ser\u00a0\u00a0 Thr\u00a0 Glu\u00a0\u00a0 —human c-Rel—————-
\n5’CCCCCCGGGG ATG GAC TGT CTG ACG GAC CTA CGC TCC ACC GAG ggt gcg tat aac ccg tat ata gag3′<\/p>\n

c.\u00a0 Upstream primer for human c-Rel fusion into GBT9:<\/p>\n

Xma1\u00a0\u00a0\u00a0\u00a0 Met—hc-Rel——————–
\nCCCCCCGGGG atg gcc tcc ggt gcg tat aac ccg<\/p>\n

d.\u00a0 Downstream primer for Env-Hu-cRel and Hu-c-Rel PCR amplification (reverse oligo)<\/p>\n

BamH1|STOP|———human c-Rel
\n5’CGGGATCCTCAtccaattgaaccgaggagaccagg3′<\/p>\n

References<\/strong><\/p>\n

1.\u00a0 Epinat J-C, Kazandjian D, Harkness DD, Petros S, Dave J, White DW and Gilmore TD. 2000a. Mutant envelope residues confer a transactivation function onto N-terminal sequences of the v-Rel oncoprotein. Oncogene 19:in press<\/p>\n

2.\u00a0 Kamens J and Brent R. 1991. A yeast transcription assay defines distinct Rel and Dorsal DNA recognition sequences. New Biologist 3:1005-1013<\/p>\n

3.\u00a0 Mosialos G and Gilmore TD. 1993. v-Rel and c-Rel are differentially affected by mutations at a consensus protein kinase recognition sequence. Oncogene 8:721-730<\/p>\n

4.\u00a0 Sif S, Capobianco AJ and Gilmore TD. 1993. The v-Rel oncoprotein increases expression from Sp1 site-containing promoters in chicken embryo fibroblasts. Oncogene 8:2501-2509<\/p>\n","protected":false},"excerpt":{"rendered":"

APPENDIX Epinat J-C, Dvorin EL and Gilmore TD. (2000) Envelope-dependent transactivation by the retroviral oncoprotein is required for transformation of chicken spleen cells. Oncogene 19 :3131-3137 A.\u00a0 Descriptions of plasmids used in this paper. (The source of the plasmid is indicated in parentheses.) 1.\u00a0 Plasmids for the expression of Rel proteins in yeast. BC102:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 Vector […]<\/p>\n","protected":false},"author":4258,"featured_media":0,"parent":337,"menu_order":7,"comment_status":"closed","ping_status":"open","template":"","meta":[],"_links":{"self":[{"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/564"}],"collection":[{"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/users\/4258"}],"replies":[{"embeddable":true,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/comments?post=564"}],"version-history":[{"count":2,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/564\/revisions"}],"predecessor-version":[{"id":566,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/564\/revisions\/566"}],"up":[{"embeddable":true,"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/pages\/337"}],"wp:attachment":[{"href":"https:\/\/www.bu.edu\/nf-kb\/wp-json\/wp\/v2\/media?parent=564"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}