Notes: This paper describes amplification of rat NHE1 cDNA using primers incorporating the Xho I and Xba I restriction endonuclease sites, Kozak consensus, ATG start site, and a stop codon. The resultant 1.1 kb NHE1 encoding-PCR product was digested with Xba I and Xho I and cloned into the pTNT™ Vector.  This construct was used as a template in a TNT^® T7 Quick Coupled Transcription/Translation reaction to generate substrates for an in vitro caspase-3 cleavage assay. Proteolytically cleaved fragments of NHE1 were run on a polyacrylamide gel and visualized by autoradiography. (3063)

Notes: To investigate the effect of the compound kamebakaurin (KA) on NF-κB, an NF-κB-responsive firefly luciferase vector was transfected into HeLa, Jurkat and THP-1 cells. The Luciferase Assay System was used to assay the level of NF-κB induction after treatment of cells with various concentrations of KA. To determine if KA influenced the DNA-binding activity of NF-κB, nuclear extracts of HeLa, Jurkat and THP-1 cells were prepared after preincubation with KA and stimulation of NF-κB activity. Control nuclear extracts were prepared from unstimulated p50- or RelA-overexpressed MCF-7 cells. In addition, the wildtype and DNA-binding mutant RelA and p50 (NF-κB) His-tagged proteins were translated using the TNT^® Quick Coupled Transcription/Translation System and subsequently purified. Using the Gel Shift Assay System, the NF-κB and AP1 oligos were tested for electromobility shifts with the prepared nuclear extracts or with purified wildtype and mutant proteins. Supershift studies using anti-p50 or anti-RelA antibodies were also performed. (3113)

Notes: The authors used Promega's TNT transcription/translation systems to produce ³⁵S labeled substrates for in vitro caspase cleavage assays. (2612)

Notes: To study the transcriptional control of human CYP8B1 by bile acids, the -514/+300 region of the gene was cloned into the pGL3-Basic Vector and subsequently mutagenized at both the 5´ and 3´ends of the CYP8B1 fragment.  The various reporter constructs were then transfected into HepG2 cells and the activity assessed using the Luciferase Assay System. Luminescence was determined using a Berthold Lumat LB 9501 luminometer. The Gal4/Id and VP16/MyoD provided in the CheckMate™ Mammalian Two-Hybrid System were used as positive controls for the in a two-hybrid assay that examined the interaction of SHP and HNF4alpha, genes that regulate CYP8B1.  The TNT^® Quick Coupled Transcription/Translation System was used to synthesize various receptor proteins cloned in expression plasmids. (3083)

Notes: The TNT® Coupled Reticulocyte Lysate System was used in conjunction with the Transcend™ Chemiluminescent Non-Radioactive Translation Detection System. Various truncations of the protein of interest were produced ranging from 67kDa to 19kDa to use in gel shift assays. The truncations were used to identify which region bound the oligo of interest. The in vitro translated proteins were also used with antibodies for supershifts. (0003)

Notes: The Serine-Threonine Phosphatase Assay System was used to monitor dephosphorylation of two phosphopeptides derived from the SCR homeodomain. Dephosphorylation was performed using the catalytic subunit of the PP2A protein. Authors also used pSI for cloning, protein kinase A enzyme, PKA and PKC assays using biotinylated peptides (SignaTECT® Protein Kinase assay systems), anti-β-galactosidase for Drosophila embryo staining, and TNT® Quick Coupled Transcription/Translation systems to produce mutant and wild type protein for gel shifts. (2150)

Notes: The Arabidopsis NPR1 protein is part of a systemic and inducible plant defense mechanism and may be the plant homolog to IκBα, the NF-κB regulator. In a yeast two-hybrid screen, it was found that NPR1 interacts with several TGA factors. To verify these interactions, NPR1 was expressed and biotinylated in E. coli XL1-Blue using the Pinpoint™ Xa-1 Vector. The biotinylated protein was affinity-purified from a sonicated culture using TetraLink™ Tetrameric Avidin Resin. The captured NPR1 was assayed for interaction with TGAs produced and radiolabled with ³⁵S-methionine. It was found that NPR-1 binds to TGA2 strongly and to TGA4 weakly. The TGA4 interaction was negative in the yeast two-hybrid system. A C150Y transition mutant of NPR1 did not bind TGA2. (2707)

Notes: Three different proteins ranging in size from 30kDa to 64kDa were translated in vitro using the TNT® T7 Quick Coupled Transcription/Translation System. The expressed proteins were used for in vitro interaction studies via immunoprecipitation. The ProtoBlot® System was used for colorimetric immunoblotting. (1302)

Notes: This paper uses the TNT^® T7 Quick Coupled Transcription/Translation System to generate C/EBP α, C/EBP β, and CREB for EMSAs. (0129)

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)

Notes: The authors used TNT^® T7 Quick Coupled Transcription/Translation System to generate radiolabeled c-Jun and its mutant variants. The radiolabeled c-jun was used for interaction studies with GST-tagged Retinoblastoma protein. (0618)

Notes: Various constructs prepared in pCDNA3 (Invitrogen) were expressed in the TNT® T7 Quick System in the presence of Canine Pancreatic Microsomal Membranes. The orientation of the proteins in the membranes were determined with proteinase K digests with and without Triton® X-100 present. Good detail is provided for the digests. (0587)

Notes: The glutathione peroxidase (GPX) cDNA was translated in vitro with the TNT^® T7 Quick Coupled System and compared to partially purified GPX native protein. Both were recognized by an antibody to the c-terminus of the cloned sequence. Further evidence that the proteins were identical were obtained from the Ouchterlony double immunodiffusion assay. Clones of the GPX protein were sequenced with the fmol^® DNA Sequencing System. (0368)

Notes: Various sequences of the synaptic scaffolding molecule (SCAM) were cloned into the pCI-neo Mammalian Expression Vector. Also, a myc tag was engineered into the vector along with the SCAM sequences. These sequences were expressed in vitro with the TNT^® T7 Quick Coupled Reticulocyte Lysate System. Extracts were prepared from COS-1 cells expressing either neurabin-1, SAPAP-1, SAPAP-2, SAPAP-3 or SAPAP-4. The extracts were separated by SDS-PAGE and transferred to nitrocellulose. The [³⁵S]met-labeled, myc-tagged proteins were reacted with the COS-1 cell extracts and interaction detected by autoradiography as well as immunoblotting with an anti-myc antibody. The method of this overlay assay was referenced. (1046)

Notes: The TNT^® T7 Quick Coupled Transcription/Translation System was used to translate the GHF-1 pituitary-specific homeodomain protein, peroxisome proliferator-activated receptor α (PPARa), retinoid X receptor α (RXRa), TATA-Binding protein, Transcription Factor IID and luciferase. The various proteins were used in gel shift assays and/or pull-down assays with GST-fusion proteins of either PPARa or RXRa. (0273)

Notes: The TNT^® T7 Quick Coupled Reticulocyte System was used to express the 134kDa Attractin protein. In the presence of Canine Pancreatic Microsomal Membranes (CMMs), the Attractin protein attained its wild-type 175kDa molecular weight. (1204)

Notes: The protein truncation test (PTT) was performed using TNT^® T7 Quick Coupled Transcription/Translation System. (0355)

Notes: Promega Casein Kinase II Peptide Substrate was used as a positive control substrate for in vitro phosphorylation assays with casein kinase II. Also, the TNT® T7 Quick Coupled Transcription/Translation System was used to make radiolabled casein kinase II beta for use in protein:protein interaction studies with GST-tagged CD5. (1381)

Notes: Promoter studies were performed in HeLa cells. The promoter constructs were assembled in the pGL3 Basic Vector (10µg) and cotransformed with the pRL-SV40 Vector (0.1µg) at a 10:1 ratio using the calcium phosphate method. Reporter activity was measured with the Dual-Luciferase^® Reporter Assay System. The TNT^® T7 Quick Coupled Transcription/Translation System was used to produce a positive control protein for Western blot analysis of transfected HeLa cell extracts. (0115)

Notes: TNT^® T7 Quick Coupled Transcription/Translation System was used to carry out Protein Truncation Test (PTT) assays on cloned cDNA products of PTEN/MMAC1 gene from microcell hybrid cell lines. (0485)

Notes: The authors used recombinant luciferase as substrate for refolding assay with Hsp70. Also translated HspB1 cDNA in TNT^® T7 Quick Coupled Transcription/Translation System as a control to confirm that the full length cDNA was isolated. (0505)

Notes: Both hMSH2 and hMLH1 cDNA were amplified in two overlapping segments (1.2 and1.7kb) and were transcribed and translated in vitro by use of the TNT^® T7 Quick Coupled Reticulocyte Lysate System. (0895)

Notes: Fragments of the dihydropyridine receptor were expressed in E.coli as his-tag fusion proteins. The fragments were immobilized on nickel columns and ³⁵S-Methionine-labeled ryanodine receptor fragments were passed over the column and assayed for retention. The labeled fragments of the receptor were generated with the TNT^® T7 Quick Coupled Transcription/Translation System. (0829)

Notes: The TNT^® T7 Quick Coupled Transcription/Translation System was used to produced the AP-2gamma protein for gel shift analysis. (0391)

Notes: The Altered Sites^® II in vitro Mutagenesis System was used to verify that cloned genes could be expressed prior to cRNA synthesis and in vitro translation in Xenopus oocytes. In vitro translation was performed with the TNT^® T7 Quick Coupled Reticulocyte Lysate System. (1008)