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J. Biol. Chem. 272, 26727-26733. Sequence of cDNAs encoding components of vascular actin single-stranded DNA-binding factor 2 establish identity to Purα and Purβ. 1997

Kelm, R. J., Jr., Elder, P. K., Strauch, A.R., Getz, M.J.

Notes: Single-stranded 3´-end biotinylated oligonucleotides were captured on the Streptavidin MagneSphere® Paramagnetic Particles (SA-PMPs) and incubated with heparin-agarose affinity-purified nuclear proteins. The reaction was performed under the conditions used for electrophoretic gel shifts. Proteins were eluted in 1X nonreducing SDS-PAGE sample buffer at 65°C for 3 minutes and analyzed by Southwestern blotting with 32P-labeled oligonucleotides. The ssDNA-affinity capture worked with the oligonucleotides prebound to the particles or after allowing the interactions to occur in solution prior to capture. The pCI Mammalian Expression Vector was used to express the proteins Purα (321 amino acids) and Purβ (324 amino acids) in mouse embryo-derived AKR-2B fibroblasts. Cell extracts were prepared and assayed by Southwestern blotting. The same proteins were in vitro translated with the TNT® Coupled Reticulocyte Lysate System and assayed as well. (0929)

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Meth. Enzymol. 283, 83-99. Small pool expression screening: Identification of genes involved in cell cycle control, apoptosis, and early development. 1997

Lustig, K.D., Stukenberg, P.T., McGarry, T.J., King, R.W., Cryns, V.L., Mead, P.E., Zon, L.I., Yuan, J. and Kirschner, M.W.

Notes: This methods article describes the in vitro expression cloning technique (IVEC) with detailed methods and materials descriptions. Plasmid or phage cDNA libraries were constructed in appropriate T7, T3 or SP expression vectors, and DH10B E. coli were transformed with the libraries. Plasmid minipreps were performed on pools of bacterial colonies, and the pooled cDNAs were used as templates in TNT® Coupled Reticulocyte Lysate System reactions. Proteins were radioactively labeled, and proteins that were phosphorylated or degraded during mitosis were identified by incubation of radiolabeled library pools with mitotic or interphase Xenopus extracts. Proteins were also tested as putative apoptotic caspase substrates using apoptotic and control Jurkat lymphoma cell extracts. In addition, unlabeled proteins were tested for DNA-binding activity in EMSAs. (2049)

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J. Biol. Chem. 272, 18990-18999. Specific Activation of Retinoic Acid Receptors (RARs) and Retinoid X Receptors Reveals a Unique Role for RARgamma in Induction of Differentiation and Apoptosis of S91 Melanoma Cells 1997

Spanjaard, R. A., Ikeda, M., Lee, P. J., Charpentier, B., Chin, W. W., Eberlein, T. J.

Notes: The CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS/PMS) was used to measure the proliferation of S91 cells. The TNT® Coupled Reticulocyte Lysate System was used to translate the retinoic acid receptor and the retinoid X receptor. These were then assayed in gel shifts. The recombinant human AP-1 and the AP-1 Consensus Oligonucleotides were used in the gel shift assays as well. (0360)

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Curr. Biol. 7, 338-348. Systematic identification of mitotic phosphoproteins. 1997

Stukenberg, P.T., Lustig, K.D., McGarry, T.J., King, R.W., Kuang, J., Kirschner, M.W.

Notes: This methods article describes the in vitro expression cloning technique (IVEC) with detailed methods and materials descriptions. Plasmid or phage cDNA libraries were constructed and transformed into the appropriate bacterial strains and pooled constructs were then used as templates in TNT® Coupled Reticulocyte Lysate System reactions. Proteins were radioactively labeled, and proteins that were phosphorylated or degraded during mitosis were identified by incubation of radiolabeled library pools with mitotic or interphase Xenopus extracts. Proteins were also tested as putative apoptotic caspase substrates using apoptotic and control Jurkat lymphoma cell extracts. (0305)

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J. Neurosci. 17, 6621-6628. T-type Ca2+ current properties are not modified by Ca2+ channel β subunit depletion in nodosus ganglion neurons. 1997

Lambert, R.C., Maulet, Y., Mouton, J., Beattie, R., Volsen, S., De Waard, M., Feltz, A.

Notes: The rat β1b and β4 as well as the rabbit β2a and β3 were in vitro transcribed/translated in the presence of [35S]methionine in the TNT® Coupled Reticulocyte Lysate System. The labeled proteins were analyzed for binding to an α1c subunit-GST fusion protein. (0832)

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J. Biol. Chem. 272, 26285-26294. TGT3, thyroid transcription factor I, and Sp1 elements regulate transcriptional activity of the 1.3-kilobase pair promoter of t1alpha, a lung alveolar type I cell gene 1997

Ramirez, M.I., Rishi, A.K., Cao, Y.X., Williams, M.C.

Notes: Luciferase studies were performed in SV40 T type II cells and IMR90 fibroblasts using constructs prepared in the pGL3 Basic Vector. Luciferase activity was measured with the Luciferase Assay System. The Erase-A-Base® System was used to generate deletion mutants of the promoter. The hepatocyte nuclear factor 3beta was produced in vitro with the TNT® Coupled Reticulocyte Lysate System and used for gel shift analysis. (0495)

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J. Biol. Chem. 272, 13426-13431. The polymyositis-scleroderma autoantigen interacts with the helix-loop-helix proteins E12 and E47. 1997

Kho, C.J., Huggins, G.S., Endege, W.O., Patterson, C., Jain, M.K., Lee, M.E., Haber, E.

Notes: Luciferase reporter studies were performed in NIH3T3 cells. The researchers created sort of a two-hybrid pGL2 Vector to demonstrate that two cotransfected proteins can interact and bind an enhancer element, thus increasing luc activity. Researchers expressed one protein as a GST fusion and the other as a 35S-met protein in the TNT® Coupled Reticulocyte Lysate System and showed that the 35S-protein could be pulled out of the lysate. (0937)

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J. Cell Biol. 138, 1343-1354. Tissue-specific expression of the L1 cell adhesion molecule is modulated by the neural restrictive silencer element. 1997

Kallunki, P., Edelman, G.M. and Jones, F.S.

Notes: Studies were performed in NIH 3T3 and Neuro2a cells. The pGEM®-luc Vector was used as a source of the luciferase gene to convert previous beta-Galactosidase reporter vector constructs to luciferase reporter vectors. The TnT® Coupled Reticulocyte Lysate System was used to verify that the cloned NRSF/REST protein could be expressed intact. (1566)

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J. Steroid Biochem. 62, 477-489. Transcriptional regulation of estrogen-responsive genes by non-steroidal estrogens: diosynolic and allenolic acids. 1997

Meyers, C.Y., Hisham, G.L. and Adler, S.

Notes: Full-length ER was expressed using the TNT® T7 Reticulocyte Lysate System. EMSAs were performed with modifications to reflect dependence on ligand. Binding reactions contained ER in reticulocyte lysate, unlabeled non-specific DNA and either hormone, various concentrations of tests compounds or ethanol and then labeled, specific DNA (30,000cpm) was added. (Parallel series was run with E2 to determine maximal binding.) The assay accurately distinguishes between compounds that do or do not act as ligands, and it allowed relative binding affinity values to be assigned for the test compound. (1842)

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Biol. Reprod. 57, 1285-92. Transcriptional regulation of human placental corticotropin-releasing factor by prostaglandins and estradiol. 1997

Dibbs, K.I., Anteby, E., Mallon, M.A., Sadovsky, Y. and Adler, S.

Notes: Estrogen receptor (ER) was expressed in the TNT® T7 Coupled Reticulocyte Lysate System. EMSAs were performed to test whether ER can bind to corticotropin-releasing factor (CRF) promoter half-sites. The in vitro translated ER was tested for binding to CRF promoter half-site oligonucleotides: The data do not support an interaction between ER and the CRF promoter half-sites. (1799)

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Diabetes 46, 1270-1275. Value of antibodies to islet protein tyrosine phosphatase-like molecule in predicting type 1 diabetes. 1997

Hawa, M., Rowe, R., Lan, M.S., Notkins, A.L., Pozzilli, Christie, M.R. and Leslie, R.D.G.

Notes: Sixty patients were screened for autoantibodies to IA-2 (a protein tyrosine phosphatase-like molecule), IA-2ic (the intracellular fragment of IA-2) and GAD65 (glutamic acid decarboxylase) using RIAs. Full-length human cDNAs were cloned into the pGEM®-4Z Vector and expressed in the TNT® Coupled Reticulocyte Lysate System in the presence of [35S]methionine. Incorporated radiolabel was determined by precipitation and scintillation counting. Immunoprecipitation was performed with 5µl of serum and counted on a multiwell counter. Antibodies to IA-2 or GAD65 were detected in 87% of newly diagnosed type 1 diabetic patients and in all 56 prediabetic samples from 11 twins. The frequencies of antibodies to IA-2ic were similar in type 1 diabetics. (1763)

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Blood 90(7), 2680-2689. Wiskott-Aldrich syndrome/X-linked thrombocytopenia: WASP gene mutations, protein expression, and phenotype. 1997

Zhu, Q., Watanabe, C., Liu, T., Hollenbaugh, D., Blaese, R.M., Kanner, S.B., Aruffo, A. and Ochs, H.D.

Notes: The system was used to isolate poly A+ RNA from the total RNA of 12 different human cell lines using the PolyATtract® mRNA Isolation System. The isolate RNA was used for Northern analysis. The fmol® DNA Cycle Sequencing System and TNT® Coupled Reticulocyte Lysate System were also used in the study. (1651)

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EMBO J. 16, 3935-3943. ZEB, a vertebrate homolog of Drosophila Zfh-1, is a negative regulator of muscle differentiation. 1997

Postigo, A.A., Dean, D.C.

Notes: Both ZEB and the DNA-binding domain of ZEB were subcloned into Promega's pCI-neo Mammalian Expression Vector just behind a FLAG epitope. After cotransfection with reporter vectors, the effect of the expressed protein was assessed in 10T1/2 fibroblasts and C2C12 cells. The two protein inserts were subcloned into a T3 RNA polymerase binding site bearing plasmid and the flag-tagged proteins were expressed in vitro with the TNT® Coupled Reticulocyte Lysate System. (0542)

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Nucl. Acids Res. 24, 1173-1174. A 5' untranslated region which directs accurate and robust translation by prokaryotic and mammalian ribosomes. 1996

Al Qahtani, A. and Mensa-Wilmont, K.

Notes: The authors compared translation of proteins with and without an RBS-hybrid-spacer from Ner. Transcripts that contained the RBS-hybrid-spacer were robustly translated, whereas transcripts with the LacZ RBS spacer were inefficiently translated. Translation was performed using the TNT® T7 Coupled Reticulocyte Lysate System (2219)

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Eur. J. Biochem. 239, 1-7. A human peroxisome-proliferator-activated receptor-g is activated by inducers of adipogenesis, including thiazolidinedione drugs. 1996

Lambe, K.G. and Tugwood, J.D.

Notes: Human PPAR-gamma and retinoid-x-receptor (RXRalpha) were expressed in the TNT® Coupled Reticulocyte Lysate System. The proteins were assessed for binding to double-stranded oligonucleotides with repeats of the sequence TGACCT, separated by 0-5 nucleotides or the ACO sequence by EMSAs. The hPPARalpha recognizes several direct repeat elements, and bound to the different oligonucleotides in a heterodimer with RXRalpha. (1806)

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Hum. Mol. Genet. 5, 675-684. A member of the MAP kinase phosphatase gene family in mouse containing a complex trinucleotide repeat in the coding region. 1996

Theodosiou, A.M., Rodrigues, N.R., Nesbit, M.A., Ambrose, H.J., Paterson, H., McLellan Arnold, E., Boyd, Y., Leversha, M.A., Owen, N., Blake, D.J., Ashworth, A., Davies, K.E.

Notes: A novel mouse gene showing high homology to the MAP kinase phosphatase gene family was cloned, and the cDNA was transcribed and translated using the TNT® SP6 Coupled Reticulocyte Lysate System with [35S]methionine. The gene produced a protein of approximately the predicted size, but the protein had slightly aberrant migration of the protein, possibly due to polyglycine or polyserine stretches and/or the proline-content of the protein. (0258)

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J. Biol. Chem. 271(4), 2271-2278. A novel meprin b' mRNA in mouse embryonal and human colon carcinoma cells. 1996

Dietrich, J.M., Jiang, W. and Bond, J.S.

Notes: Both the b and b' meprin isoforms were synthesized in the presence or absence of the microsomes. The ~70kDa protein was shifted to ~110kDa in the presence of the membranes. (2046)

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Cell Signal 8, 43-53. Altered Gs alpha N-terminus affects Gs activity and interaction with the G beta gamma subunit complex in cell membranes but not in solution. 1996

Warner, D. R. , Okuya, S. , Rebois, R. V.

Notes: Recombinant Gs and adenylyl cyclase (ACIV) proteins were produced using the TNT® T7 Coupled Reticulocyte Lysate System. The Gs alpha proteins were altered in their N-termini and shown to have altered activity. Both Gs alpha52 and Gs alpha54 can form a heterotrimer with G beta gamma and could activate adenylylcyclase. When incorporated into membranes, Gs alpha54 retained these abilities. (0168)

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J. Biol. Chem. 271, 20145-21050. Alternative exon splicing controls a translational switch from activator to repressor isoforms of transcription factor CREB during spermatogenesis. 1996

Walker, W.H., Girardet, C. and Habener, J.F.

Notes: CREB was transcribed and translated in the TNT® T7/SP6 Coupled Reticulocyte Lysate System in the presence of [35S]methionine. CREB protein was tested for binding to a consensus CRE. DNA-CREB complexes were formed corresponding to homodimers of the I-CREB(l) and I-CREB(s) as well as heterodimers among the two I-CREB, full-length activator CREBs and truncated CREB. (1820)

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Am. J. Physiol. 271, C825-C832. Amino-terminal processing of the catalytic subunit from Na(+)-K(+)-ATPase. 1996

Pressley, T. A., Allen, J. C., Clarke, C. H., Odebunmi, T., Higham, S. C.

Notes: The catalytic subunit from the Na+-K+-ATPase was expressed in Wheat Germ Extracts or Rabbit Reticulocyte Lysates. In immunoblots using an antibody that is specific to the first 9 residues of the predicted protein, the in vitro synthesized protein but not the in vivo synthesized protein was detected, suggesting that the protein is processed in vivo. Processing was inefficient or absent in the TNT® Coupled Reticulocyte Lysate Systems. (0546)

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Mol. Endocrinol. 96, 879. An alternate spliced polycistronic mRNA encoding cyclic adenosine 3´,5´-monophosphate (cAMP)-responsive element-CREB (cAMP response element binding protein) in human testis extinguishes expression of an internally translated inhibitor CREB isoform. 1996

Girardet, C., Walker, W.H. and Habener, J.F.

Notes: DNA binding was examined using EMSAs. Proteins encoded by CREB, CREB-W and CREB-WZ were expressed in TNT® T7 or SP6 Reticulocyte Lysate Systems. Extracts were incubated with a 32P-labeled CRE-containing oligo. CREB produced a single complex with the CRE probe, and CREB-W and CREB-WZ produced several smaller complexes. (1801)

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FEBS Lett. 382, 265-270. Anti-head and anti-tail antibodies against distinct epitopes in the catalytic subunit of protein kinase A. Use in the study of the kinase splitting membranal proteinase KSMP. 1996

Chestukhin, A., Litovchick, L., Batkin, M. and Shaltiel, S.

Notes: Wild type and mutant versions of the catalytic subunit (C) of bovine protein kinase A were expressed in the TNT® Coupled Reticulocyte Lysate System. Immunoprecipitation of the in vitro translated proteins verified the specificity of alphaP-2 antibodies. (1762)

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Int. J. Cancer 68, 305-312. APC gene mutations and allelic losses in sporadic ampullary tumours: Evidence of genetic difference from tumours associated with familial adenomatous polyposis. 1996

Achille, A., Scupoli, M.T., Magalini, A.R., Zamboni, G., Romanelli, M.G., Orlandini, S., Biasi, M.O., Lemoine, N.R., Accolla, R.S. and Scarpa, A.

Notes: Eighteen sporadic neoplasms of ampullary origin were examined for the involvement of mutations in APC, Ras, p53 and chromosomal 5q21 allelic losses. 5q21 loss (including APC) occurred in 50% and APC mutations occurred in 17% of the ampullary tumors. 5q21 loss and mutations in APC and Ras occur in early tumor development, and p53 inactivation is associated with progression of malignancy. The researchers used PTT for APC mutation detection in the TNT® T7 Coupled Reticulocyte Lysate System with chemiluminescent labeling instead of radiolabeling of proteins. (2056)

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J. Clin. Endocrinol. Metab. 81, 700-706. Binding of antithyrotropin receptor autoantibodies in Graves’ Disease serum to nascent, in vitro translated thyrotropin receptor: ability to map epitopes recognized by antibodies. 1996

Morgenthaler, N.G., Tremble, J., Huang, G., Scherbaum, W.A., McGregor, A.M. and Banga, J.P.

Notes: The full-length and extracellular region of the human TSH-R (TSH-R and TSH-R.E, respectively) were expressed in the TNT® Coupled Reticulocyte Lysate System in the presence of [35S]methionine. First, immunoprecipitation was performed to the proteins with one rabbit polyclonal antiserum and three murine antibodies that recognize 3 different determinants on the proteins. From the full-length receptor, the antibodies recognize the 97kDa as well as other smaller bands. The TSH-R was also immunoprecipitated with sera from Graves’ Disease patients. The majority of sera precipitated the TSH-R 97kDa and TSH-R.E 50kDa proteins, with some sera precipitating other, smaller proteins. There was variability in strength between different samples. (1773)

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J. Med. Genet. 33, 721-725. BRCA1 mutations in a selected series of breast/ovarian cancer patients. 1996

Garvin, A. M. , Spycher, M. , Haner, M. , Torhorst, J. , Muller, H. , Herrmann, R. , Rochlitz, C. , Weber, W. , Scott, R. J.

Notes: PTT was used to rapidly screen the BRCA1 gene. Transcription and translation was performed on five overlapping fragments (amplified by PCR) of the entire coding region of the BRCA1 cDNA or genomic DNA using the TNT® T7 Coupled Reticulocyte Lysate System. (1144)

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