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J. Gen. Virol. 80, 1293-1303. Domain mapping of the human cytomegalovirus IE1-72 and cellular p107 protein-protein interaction and the possible functional consequences. 1999

Johnson, R.A., Yurochko, A.D., Poma, E.E., Zhu, L., Huang, E.-S.

Notes: SAOS-2 human osteosarcoma cells transfected with either a p107 expression vector or vector only were challenged with HCMV, and the proliferation was measured with the CellTiter 96® Non-Radioactive Cell Proliferation Assay. The TNT® Coupled Reticulocyte Lysate System was used to demonstrate the interaction of the HCMV protein IE1-72 and the p107 protein. (0942)

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Plant Cell 11(6), 1129-1140. Effect of pectin methylesterase gene expression on pea root development. 1999

Wen, F. , Zhu, Y. , and Hawes, M. C.

Notes: The cDNA of the pectin methyltransferase was translated in vitro with the TNT® Coupled Reticulocyte Lysate System in the presence of the Transcend™ Biotinylated tRNA. The protein was used for in vitro enzymatic assay of activity. The assay was colorimetric and the heme in the rabbit lysate interfered. The hemoglobin was removed by acid precipitation and the method is referenced. The enzyme contains 555 amino acids and 39 lysines. (0029)

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Proc. Natl. Acad. Sci. USA 96, 8651-8656. Elevated retinoic acid receptor β4 protein in human breast tumor cells with nuclear and cytoplasmic localization. 1999

Sommer, K.M., Chen, L.I., Treuting, P.M., Smith, L.T., Swisshelm, K.

Notes: Clones of the retinoic acid receptors were subcloned in the the pSP64 Poly(A) Vector and translated in vitro with the TNT® SP6 Reticulocyte Lysate System. The expressed proteins were confirmed as retinoic acid receptors by Western blotting. (0352)

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Genetics 151, 617-631. Genetic and molecular analysis of fox-1, a numerator element involved in Caenorhabditis elegans primary sex determination. 1999

Skipper, M., Milne, C. A., Hodgkin, J.

Notes: TNT® Coupled Reticulocyte Lysate System was used in in vitro transcription/translation reactions to produce 35S Met-labeled FOX-1 and luciferase protein for RNA binding assay. (0375)

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J. Biol. Chem. 274, 9029-9037. Mutant vasopressin precursors that cause autosomal dominant neurohypophyseal diabetes isipidus retain dimerization and impair the secretion of wild-type proteins. 1999

Ito, M., Yu, R.N., Jameson, J.L.

Notes: Wildtype arginine vasopressin and wildtype arginine vasopressin with a Myc-His tag were separately translated in vitro with the TNT® Coupled Reticulocyte Lysate System in the presence of Canine Pancreatic Microsomal Membranes (CMMs). The translated proteins were allowed to interact and the complex was brought down via the His tag. (0962)

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J. Biol. Chem. 274, 3151-3158. Pituitary tumor-transforming gene protein associates with ribosomal protein S10 and a novel human homologue of DnaJ in testicular cells. 1999

Pei, L.

Notes: The authors used the TNT® Coupled Reticulocyte Lysate System with Transcend™ Biotinylated tRNA to synthesize S10 and HSJ2 protein for protein:protein interaction studies with GST-tagged PTTG. (0556)

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J. Cell Biol. 145, 237-254. RAE1 is a shuttling mRNA export factor that binds to a GLEBS-like NUP98 motif at the nuclear pore complex through multiple domains. 1999

Pritchard, C.E.J., Fornerod, M., Kasper, L.H., van Deursen, J.M.A.

Notes: The cDNA for the 45kDa HA-tagged RAE1 protein was subcloned into the pSP73 Vector and expressed in the TNT® Reticulocyte Lysate System. The labeled protein was used demonstrate protein-protein interactions. (2188)

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J. Biol. Chem. 274, 15671-15677. Splice variants of intersectin are components of the endocytic machinery in neurons and nonneuronal cells. 1999

Hussain, N.K., Yamabhai, M., Ramjaun, A.R., Guy, A.M., Baranes, D., O'Bryan, J.P., Der, C.J., Kay, B.K., McPherson, P.S.

Notes: The proteins Ibp2, MP90 and luciferase were translated in vitro with the TNT® Coupled Reticulocyte Lysate System in the presence of the Transcend™ Biotinylated tRNA. The translated proteins were reacted with a glutathione bead-immobilized portion of the clathrin heavy chain. Bound proteins were solubilized in SDS sample buffer and the proteins detected by Western detection with the Transcend™ Non-Radioactive Translation Detection System. (0990)

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J. Biol. Chem. 274, 14352-14358. The autonomous transactivation domain in helix H3 of the vitamin D receptor is required for transactivation and coactivator interaction. 1999

Kraichely, D.M., Collins, J.J. III, DeLisle, R.K. , MacDonald, P.N.

Notes: The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to introduce a variety of different point mutations into the vitamin D receptor. The mutant proteins were also expressed in vitro with the TNT® Coupled Reticulocyte Lysate System and used for in vitro protein interaction assays. (0889)

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Mol. Cell 4, 573-584. The balance between two isoforms of the Drosophila RNA-binding protein How controls tendon cell differentiation. 1999

Nabel-Rosen, H., Dorevitch, N., Reuveny, A., Volk, T.

Notes: The Streptavidin MagneSphere® Paramagnetic Particles (SA-PMPs) were used to capture RNA:RNA binding protein complexes formed with biotinylated RNA and tissue extracts. The captured complexes were boiled in SDS sample buffer, electrophoresed and blotted for detection. The results of the capture were also confirmed by in vitro translation of the protein of interest with the TNT® Coupled Reticulocyte Lysate System and doing the same capture. (0639)

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Am. J. Hum. Genet. 65, 1725-1732. The genetic epidemiology of early-onset epithelial ovarian cancer: a population-based study. 1999

Stratton, J. F., Thompson, D., Bobrow, L., Dalal, N., Gore, M., Bishop, D. T., Scott, I., Evans, G., Daly, P., Easton, D. F., Ponder, B. A.

Notes: BRCA2 was screened for mutations in the ovarian cancer cluster region by means of the protein-truncation test (PTT). PTT was performed with the TNT® Coupled Reticulocyte Lysate System, incorporating [35S] methionine for protein detection. (0343)

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Mol. Cell. Biol. 20, 900. The Homeodomain of PDX-1 Mediates Multiple Protein-Protein Interactions in the Formation of a Transcriptional Activation Complex on the Insulin Promoter 1999

Kinuko Ohneda, Raghavendra G. Mirmira, Juehu Wang, Jefrey D. Johnson, and Michael S. German

Notes: The authors use the Luciferase Assay System to measure luciferase activity in mammalian and insect cells 40-48 hours after transfection. The TNT® T7 Coupled Reticulocyte Lysate System is used to generate in vitro translated and [35S]methionine-labeled proteins for in vitro protein-protein interaction assays (2412)

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Genetics 153, 1561-1572. The identification of Wos2, a p23 homologue that interacts with Wee1 and Cdc2 in the mitotic control of fission yeasts. 1999

Munoz, M.J., Bejarano, E.R., Daga, R.R., Jimenez, J.

Notes: For in vitro experiments, 35S-methionine-labeled proteins were synthesized using the TNT® Coupled Reticulocyte Lysate system. Immunoprecipitation was performed and the labeled proteins were resolved by electrophoresis in polyacrylamide gels and visualized by autoradiography. (0672)

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Genetics 151, 45-55. The MSN1 and NHP6A genes suppress SWI6 defects in Saccharomyces cerevisiae. 1999

Sidorova, J., Breeden, L.

Notes: In vitro translation of Swi4 was performed by use of TNT® Coupled Reticulocyte Lysate System along with recombinant Swi6 purified from E. coli to study protein-protein and protein-DNA interaction. (0406)

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Cell 93, 215-228. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy 1998

Molkentin, J.D., Lu, J.-R., Antos, C.L., Markham, B., Richardson, J., Robbins, J., Grant, S.R. and Olson, E.N.

Notes: The NF-AT3 transcription factor was identified as a GATA4-interacting factor in a yeast two-hybrid screen. This interaction results in synergistic activation of cardiac transcription. A partial NF-AT3 cDNA containing a Rel homology domain (RHD), along with a Flag epitope tag, was expressed in the presence and absence of [35S]methionine using the TNT® Coupled Reticulocyte Lysate System. The proteins were immunoprecipitated with the Flag antibody and the Rel homology domain of the NF-AT3. The NF-AT3 RHD was also expressed in the TNT® System without label and used in gel mobility shift assays with ds-oligonucleotides corresponding to sequence within the BNP promoter. To demonstrate specificity of the binding reaction, site-directed mutations were generated in the promoter sequences, and the promoter sequences were tested for sensitivity to NF-AT3. (1844)

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Cell 92, 725-734. ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. 1998

Zhang, Y., Xiong, Y. and Yarbrough, W.G.

Notes: A yeast two-hybrid screen was used to search for proteins that physically interact with human ARF-GAL4 binding domain fusions. A truncated MDM2 protein interacted with the ARF fusion protein. This interaction was confirmed by expressing the proteins either together or separately in the TNT® Reticulocyte Lysate System and mixing the translation reactions. After incubation, the mixtures were treated with NP-40 lysis buffer, immunoprecipitated with antibodies to ARF, MDM2 or myc and resolved by SDS-PAGE. MDM2 coprecipitated with ARF using either anti-ARF or anti-myc antibodies, and ARF was detected when immunoprecipitated with anti-MDM2 antibodies. (1782)

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Proc. Natl. Acad. Sci. USA 95, 8058-8063. Basal and human papillomavirus E6 oncoprotein-induced degradation of Myc protein by the ubiquitin pathway. 1998

Gross-Mesilaty, S., Reinstein, E., Bercovich, B, Tobias, K.D., Schwartz, A.L., Kahana, C., Ciechanover, A.

Notes: The pCI-neo Mammalian Expression Vector was used to express human c-myc in Cos cells. The plasmid was also used as a template in TNT® Coupled Wheat Germ System for in vitro studies. Both myc proteins and E6 proteins were expressed in vitro using the TNT® Coupled Reticulocyte Lysate System for in vitro studies. Proteins expressed in the in vitro system were partially purified over a DEAE column prior to being assayed. (1077)

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J. Biol. Chem. 273, 13918-13924.. Binding of hsp90 to the glucocorticoid receptor requires a specific 7-amino acid sequence at the amino terminus of the hormone-binding domain. 1998

Xu, M., Dittmar, K.D., Giannoukos, G., Pratt, W.B., Simons, Jr., S.S.

Notes: The GeneEditor™ Site-Directed Mutagenesis System was used to make a cysteine to proline mutation in a mammalian expression construct. The mutation was introduced with the aid of a 25-mer oligonucleotide. To aid in screening for mutants, a unique Pvu II site was also introduced, and the TNT® Coupled Reticulocyte Lysate System was used to confirm some protein-protein interactions.. (0124)

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J. Biol. Chem. 273, 8369-8375. Biochemical function of mouse minichromosome maintenance 2 protein. 1998

Ishimi, Y., Komamura, Y., You, Z., Kimura, H.

Notes: Mouse minichromosome maintenance 2 protein (MCM2) synthesized in vitro using the TNT® Coupled Reticulocyte Lysate System was used in histone binding studies. Tfx™-20 Reagent was used to transfect HeLa cells with truncations of the MCM2 gene (2:1 lipid:DNA). (1005)

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J. Biol. Chem. 273, 28746-28751. Carboxylmethylation of the beta subunit of xENaC regulates channel activity. 1998

Rokaw, M.D., Wang, J.M., Edinger, R.S., Weisz, O.A., Hui, D., Middleton, P., Shlyonsky, V., Berdiev, B.K., Ismailov, I., Eaton, D.C., Benos, D.J., Johnson, J.P.

Notes: The TNT® T7 Coupled Reticulocyte Lysate System was used to synthesize alpha, beta and gamma subunits of the epithelial sodium channel from A6 cells. These proteins were then carboxymethylated using methyltransferase prepared from the cell membranes of A6 cells pretreated with aldosterone. The in vitro translated subunits were reconstituted in lipid bilayers and subjected to methylation to study the effect of carboxymethylation on activity of the sodium channel. The authors also used Promega's Immobilized Anti-Chicken IgY to immunoprecipitate the beta and gamma subunits from A6 cell membranes using IgY antibodies to these proteins. (0494)

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J. Biol. Chem. 273, 9158–9167. Caspase cleavage of gene products associated with triplet expansion disorders generates truncated fragments containing the polyglutamine tract. 1998

Wellington, C.L., Ellerby, L.M., Hackam, A.S., Margolis, R.L., Trifiro, M.A., Singaraja, R., McCutcheon, K., Salvesen, G.S., Propp, S.S., Bromm, M., Rowland, K.J., Zhang, T., Rasper, D., Roy, S., Thornberry, N., Pinsky, L., Kakizuka, A., Ross, C.A., Nicholson, D.W., Bredesen, D.E., and Hayden, M.R.

Notes: The TNT® Coupled Wheat Germ Extract and the TNT® Coupled Rabbit Reticulocyte Lysate Systems were used to in vitro express and 35S-methionine label various Huntington protein mutants and polyglutamine-containing proteins. These proteins included a full-length 210KDa Huntington, a 210KDa Atrophin-1, and a 140KDa Androgen Receptor protein. The expressed proteins were incubated with apoptotic extracts or purified caspases to detect cleavage by caspases. Mutated Huntington constructs were cloned into the pCI-neo Vector.  Data from autoradiographs were quantitated by laser band densitometry. (3019)

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J. Biol. Chem. 273, 33580-33587. Characterization of the Ah receptor-associated protein, ARA9. 1998

Carver, L. A. , LaPres, J. J. , Jain, S. , Dunham, E. E. , Bradfield, C. A.

Notes: The authors used the TNT® Coupled Reticulocyte Lysate System to synthesize ARA9 and deletions thereof for binding studies with AHR. (1350)

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J. Biol. Chem. 273, 23469-23475. Cloning and deduced amino acid sequence of a novel cartilage protein (CILP) identifies a proform including a nucleotide pyrophosphohydrolase. 1998

Lorenzo, P., Neame, P., Sommarin, Y. and Heinegård, D.

Notes: A novel furin-like human articular cartilage, cartilage intermediate layer protein (CILP), was expressed in the TNT® T3 Coupled Reticulocyte Lysate System. The CILP cDNA was cloned into a pBluescript II KS(1) vector. The researchers used 500ng of the plasmid in the in vitro transcription/translation reaction.  Reactions were supplemented with  [35S]methionine and 20 units RNasin® Ribonuclease Inhibitor. The reaction products were ethanol precipitated and resuspended in SDS loading buffer before loading on a SDS-PAGE gel for visualization. (2687)

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J. Biol. Chem. 273, 35201-35207. Degradation of proto-oncoprotein c-Rel by the ubiquitin-proteasome pathway. 1998

Chen, E. , Hrdlickova, R. , Nehyba, J. , Longo, D. L. , Bose, H. R. Jr, Li, C. C.

Notes: C-terminally truncated versions of c-rel were synthesized using the TNT® Coupled Reticulocyte Lysate System. Proteins were purified away from unincorporated methionine using Centricon-30 ultrafiltration membranes. The pCI-neo Mammalian Expression Vector was used to express chicken c-rel in NIH3T3 and COS cells. (1322)

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Biochemistry 37, 10134-10143. DNaseY: a rat DNaseI-like gene coding for a constitutively expressed chromatin-bound endonuclease. 1998

Liu, Q.Y., Pandey, S., Singh, R.K., Lin, W., Ribecco, M., Borowy Borowski, H., Smith, B., LeBlanc, J., Walker, P.R., Sikorska, M.

Notes: The authors used the PolyATtract® mRNA Isolation System IV to isolate mRNA from total RNA and the Wizard® PCR Preps DNA Purification Resin to purify a first-strand cDNA synthesis product.  TNT® T7 Coupled in vitro Transcription/Translation Reticulocyte Lysate System was used to synthesize radiolabeled DNaseY. And to test for signal peptide function they added Canine Pancreatic Microsomal Membranes (CMMs). (0779)

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