Notes: [³⁵S]methionine-labeled AMPK (AMP activated protein kinase) subunits were cotranslated in vitro using the TNT^® Coupled Reticulocyte Lysate System. The proteins were co-immunoprecipitated and shown to associate in vitro. The results indicate that a ternary complex is formed between the alpha, beta and gamma subunits rather than a mixture of dimers. When the lysates were programmed without beta, the alpha and gamma did not co-immunoprecipitate. (1779)

Notes: The IAR PTP protein (1015 residues) and the B11 protein (820 residues) were synthesized in the presence or absence of the Canine Microsomal Membranes. The predicted protein sequences contain one transmembrane domain. Proteinase K digestion in the presence or absence of Triton X-100 demonstrated the protection of a fragment consistent with the single membrane spanning domain. (1616)

Notes: cDNAs were synthesized from rat prostate total RNA using primers to the most conserved regions of the DNA-binding domain and ligand-binding domain of the nuclear receptor family. Protein was synthesized from one clone using the TNT^® Coupled Reticulocyte Lysate System and incubated with [³H]-labeled estradiol (E2) and various competitors. Only estrogens competed effectively with E2; therefore, the protein is probably an estrogen receptor. (0858)

Notes: [³⁵S]methionine-labeled cofactor A was produced using the TNT^® T7 Coupled Reticulocyte Lysate System. The protein interacts with beta-tubulin monomer on a Superose^® 6 gel-filtration column. In addition, Cofactor A binds native or quasinative beta-tubulin in folding buffer. (1785)

Notes: The VDR protein was translated in the TNT^® Coupled Reticulocyte Lysate System in the presence of [³⁵S]methionine. The protein was incubated with various concentrations of hormones (1,25-(OH)2 vitamin D3, OCT, KH1060, all-trans retinoic acid, progesterone, 17β-estradiol, thyroid hormone, and 9-cis retinoic acid) and then exposed to trypsin, chymotrypsin and proteinase K. (0215)

Notes: PCR was performed using primers to introduce a T7 promoter and a translation initiation sequence into PCR products used as templates in TNT^® Coupled Reticulocyte Lysate System reactions. Truncation mutations were detected in 12/26 FAP patients; however, 33% of confirmed protein truncations in APC were not detected using the protein truncation test (PTT). (1238)

Notes: Mutations were generated in the amphipathic helix of CBV3 protein 2B. RNA transcripts of CBV3 protein 2B were synthesized and translated in TNT® T7 Coupled Reticulocyte Lysate System supplemented with 20% HeLa cell initiation factors, to see if mutations affected polyprotein synthesis and processing. None of the mutations affected polyprotein processing. (1918)

Notes: The TNT^® Coupled Reticulocyte Lysate System was used to produce [³⁵S]methionine-labeled CR16 protein. The labeled protein was incubated with a GST-fusion of SH3 immobilized on glutathione agarose and a directed interaction was determined. (1534)

Notes: The TNT^® Coupled Reticulocyte Lysate System used an alternative initiation codon when translating 5´ deletion mutants of the human D4 receptor. This result suggests that an amino-terminal truncated D4 receptor might exist in vivo and that the amino terminus may stabilize the active state of the receptor. (0415)

Notes: Wild type and mutant D4-GDI were expressed in the TNT^® T7 Coupled Reticulocyte Lysate System and labeled with [³⁵S]methionine. The protein was then incubated with bacterially expressed CPP32, the apoptosis protease. The wild type protein was cleaved into a fragment of the same size as that seen in vivo, but an Asp19 to Asn mutation in D4-GDI blocked the cleavage event. (2041)

Notes: Chimeric in-frame fusion proteins of the N-terminal portion of different mutants of RD1 and the N-terminal region of CAT were produced using the TNT^® SP6 Coupled Reticulocyte Lysate System. The chimeric protein was added to COS cell membranes and checked for membrane association. This work, coupled with structural studies, allowed for the identification of the N-terminal splice region that confers membrane targeting upon RD1. (1787)

Notes: TCF-1 proteins were produced using the TNT^® SP6 Coupled Reticulocyte Lysate System. TCF-1 proteins were used in gel retardation assays, which showed that the extended TCF-1 N-terminus does not reconstitute an intramolecular DNA-binding inhibition domain. (1817)

Notes: Nine germline mutations in BRCA1 were identified by use of the SSCP and heteroduplex analysis (HA) and by the PTT of exon 11 of BRCA1. PTT was performed using the TNT^® T7 Coupled Reticulocyte Lysate System. Six of the truncation mutations were detected by PTT including two cases that were not detected by HA. (0984)

Notes: The combination of RT-PCR and PTT were used to detect novel mutations in 4/6 DMD patients. The PTT analysis was performed using the TNT^® T7 Coupled Reticulocyte Lysate System. (0243)

Notes: AHR and ARNT, as well as their mutant derivatives, were expressed in the TNT^® T7 Coupled Reticulocyte Lysate System in the presence or the absence of [³⁵S]methionine. The radiolabeled AHR and ARNT were mixed with equimolar amounts of mutant ARNT and AHR, respectively. Immunoprecipitation was performed, and all AHR mutants dimerized with ARNT as efficiently as wild type AHR. (1758)

Notes: C-terminal and N-terminal GT-2 deletion mutant proteins as well as site-directed GT-2 mutant proteins were synthesized from PCR templates using the TNT^® T7 Coupled Reticulocyte Lysate System. Polypeptide segments in the N-terminal and C-terminal domains of GT-2 were analyzed for differential DNA binding to GT-box target sites. (0609)

Notes: In vitro translated Kir protein was produced from the TNT^® Coupled Reticulocyte Lysate System and used to verify antibody detection and specificity in Western blots. (0242)

Notes: [³⁵S]labeled FR-19B (FGF-regulated 19B) protein was transcribed using the T7 RNA polymerase and translated in the TNT^® Coupled Reticulocyte Lysate System. A gel shift assay was performed with these proteins and double-stranded oligo from the SV40 enhancer. The results indicate that FR-19B specifically binds to the GT-IIC motif. (1804)

Notes: SAPK proteins were synthesized using the TNT^® T3 Coupled Reticulocyte Lysate System. These proteins were incubated with GST-cJun or GST-ATFa fusion proteins. The results demonstrate that the SAPKs interact with ATFa and c-Jun in vitro. (1749)

Notes: SSCP and heteroduplex analysis were used to screen exons of APC from gastric cancer lines; in addition, protein truncation test (PTT) was used to screen the MCR (mutation cluster region) of exon 15. The template was produced by PCR of genomic MCR DNA and used in the TNT^® Coupled Reticulocyte Lysate System in the presence of ³⁵S methionine. (0309)

Notes: The MyoD kinase was translated using the TNT^® Coupled Reticulocyte Lysate System in the presence or absence of the microsomes. No change in mobility was noted for the MyoD kinase but the a-factor control did get glycosylated. The MyoD kinase did not gain protection from proteinase K but the a-factor was fully protected. (1604)

Notes: The protein truncation test (PTT) was used to identify translational stops in transcripts of the APC (adenomatous polyposis coli) gene from familial adenomatous polyposis patients. Overlapping sets of oligonucleotide primers were used to amplify specific regions of APC cDNAs. These gene fragments were expressed in the TNT^® T7 Coupled Reticulocyte System. (1454)

Notes: Native and mutated AR cDNAs were transcribed with the TNT® T7 Coupled Reticulocyte Lysate System. The reaction mixtures were divided and supplemented with either androgen or vehicle. For EMSAs, the preincubated receptors were placed in binding reactions with [32P]-labeled dsDNA corresponding to a glucocorticoid/androgen-responsive element (ARE). All AR receptors with intact DNA-binding domains formed specific complexes with the ARE. The presence of active hormone altered the mobility of receptor-DNA complexes probably due to a conformational change. Mutant proteins could form heterodimers with native AR that interacted with DNA. Androgen receptor (AR) was translated using the Rabbit Reticulocyte Lysate System, and RelA was translated using Wheat Germ Extract. Coimmunoprecipitation was performed with anti-AR or anti-RelA, but no specific association between AR and RelA was detected (even with the cross-linking agent dithiobis(succinimidylpropionate) present). This finding does not exclude the possibility that AR-RelA interactions occur in vivo. (1669)

Notes: RRC3 constructs with an HA epitope ligated to the C-terminal end were in vitro transcribed and translated using the TNT^® T7 Coupled Reticulocyte Lysate System. The proteins were immunoprecipitated with mouse anti-HA. Stability of the different RRC3 proteins was examined by mixing transcription/translation reactions with cytosol. Proteins from all constructs were equally stable with little degradation. (1759)

Notes: Mouse peroxisome proliferator-activated receptor alpha (PPARalpha) and retinoid X receptor (RXRalpha) were synthesized in vitro with the TNT® Coupled Reticulocyte Lysate System. Gel shifts were performed using [32P]labeled acyl-CoA oxidase oligo. PPARalpha and RXRalpha bind the AOX-PPRE as a heterodimer, but neither binds the AOX-PPRE alone. PPARalpha inhibits TRbeta/RXRalpha heterodimer formation on a DR+4 T3 response element. (1815)