Notes: Vascular cell adhesion molecule 1 (VCAM1) is associated with several chronic inflammatory conditions. CAM741 is a fungus-derived cyclopeptide that inhibits VCAM1 expression in endothelial cells. This study investigated the mechanism by which this inhibition occurs. HEK293 cells transfected with a VCAM1 expression vector and exposed to CAM741 expressed fully glycosylated VCAM1, indicating that the inhibitor compound did not affect protein production. Production of VCAM1 in the TNT^® Coupled Rabbit Reticulocyte Lysate System with and without Canine Microsomal Membranes, and in the presence and absence of CAM741 showed that the appearance of the glycosylated form of the protein was dose-dependently inhibited in the presence of CAM741. This indicated that CAM741 inhibited translocation of VCAM1 across the membranes. The authors further localized the target to the signal peptide region of the VCAM1 protein by examining the effect of various mutations in the signal peptide and other regions of the VCAM1 protein on susceptibility to the inhibitor compound and on translocation. (3580)

Notes: The MagneGST™ Protein Purification System was used to purify GST fusion proteins of the oncoprotein HPV16 E7 or various mutants of HPV16 E7. The purified GST fusion proteins were used for in vitro binding experiments with steroid receptor coactivator 1 (SRC-1), which was produced using the TNT^® T7 Coupled Wheat Germ Extract System and labeled with the Transcend™ Non-Radioactive Translation Detection System. GST pull-down assays were resolved by Western analysis using streptavidin-horseradish peroxidase and alpha-GST. To determine the effects of endogenously expressed HPV16 E7 on SCR-1-mediated transcription, luciferase reporters under the control of either the IL-8 promoter or an artificial promoter containing three estrogen response elements repeats (3 × EREs) were cotransfected with a Renilla control vector into two human cervical cancer lines (C33A and CaSki) using either the Transfast™ Transfection Reagent or another commercial transfection reagent. The Dual-Luciferase^® Reporter Assay was then used to determine luciferase activity to functionally map the E7-interacting domain and to determine the effects of high- and low-risk PHV E7s on SRC-1-mediated transcription. (3459)

Notes: The authors examined the role of sumoylation in transcriptional repression by the homodimeric MafG protein and in transcriptional activation by a MafG/p45 heterodimer. To monitor transcription levels in the presence of MafG and mutated forms of MafG, the authors cotransfected 293T cells with a plasmid containing three copies of a Maf recognition site (MARE) driving expression of the firefly luciferase reporter gene, and an unspecified plasmid with the Renilla luciferase reporter gene for normalization. Reporter gene activities were measured using the Dual-Luciferase^® Reporter Assay System. To examine DNA binding ability, polyhistidine-tagged MafG and MafG mutants were expressed in the TNT^® Coupled Wheat Germ Extract System and used in electrophoretic mobility shift assays. The DNA-binding ability was also examined using biotinylated double-stranded DNA probes with a MARE sequence. The MafG proteins were allowed to bind to this probe, and the protein-DNA complexes were captured using the TetraLink™ Tetrameric Avidin Resin then separated by SDS-PAGE. (3660)

Notes: These authors use the TNT^® T7 Coupled Wheat Germ Extract to synthesize Chk2 that is not hyperphosphorylated, lacked Thr²⁶/Ser²⁸ and Thr⁶⁸ phosphorylation, and had minimal autophosphorylation activity. They then determined the chromatin binding properties of the resulting hypophosphorylated Chk2. (3462)

Notes: In this paper, Promega Wheat Germ Extract System was used to express the oncogene product c-Abl and a mutant (Abl-PP). These in vitro translated proteins were assayed by Western blotting and immunoprecipitation. The Dual-Luciferase^® Reporter Assay System was also used during the course of these experiments. (2493)

Notes: Lymphoid Enhancer-Binding Factor-1 (LEF-1) cDNA was cloned into the pTNT™ Vector. One microgram of the resultant construct was used as a template for in vitro transcription/translation reactions using the TNT^® T3 Coupled Wheat Germ Extract System. Western blotting and gel shift assays verified the size and function of LEF-1 expressed from the reactions.  (2721)

Notes: In this paper, proteins were expressed from PCR-generated template DNA using TNT^® Quick for PCR DNA, the TNT^® T7 Wheat Germ Extract System and the E.coli T7 S30 Extract System. (2603)

Notes: Several proteins were translated using the TNT^® Coupled Wheat Germ Extract System. p105 was expressed, then used as a substrate for ubiquitination, with the E1 protein necessary for the process contributed by the WGE. (2157)

Notes: Recombinant HNF-6 was expressed using the TNT^® Coupled Wheat Germ Extract System. The resulting protein was used in a gel shift assay, using 5µl of the reaction in a total volume of 20µl. (2156)

Notes: To clone the SUMO coupling enzyme, the SUMO peptide was attached to a column, and HeLa extracts were run through the column. Eluted proteins were separated by SDS-PAGE, stained and the appropriate bands were cut from the gel. The gel bands were then digested with an in-gel trypsinization protocol using the Sequencing-Grade Modified Trypsin. The resultant bands were separated by HPLC, sequenced and the full sequences were obtained from the ATCC database. The SUMO-1 Activating enzyme was found to contain two subunits, SAE-1 (38kDa) and SAE-2 (72kDa). These subunites were expressed in vitro using the TNT^® Coupled Wheat Germ Extract System and found to interact. (1226)

Notes: In this paper, Promega's TNT® Coupled Wheat Germ Lysate System was used to make radiolabeled HOXB7 protein, and deletion variants thereof, for protein:protein interaction studies with GST-tagged I kappa B alpha. (1363)

Notes: The TNT^® Coupled Wheat Germ Extract System was used to generate radiolabeled Cdx2/3 for protein:protein interaction studies with GST-tagged Pax-6 domains. (0479)

Notes: This paper describes use of the PolyATtract^® mRNA Isolation System, pCI Mammalian Expression Vector, and TNT^®  Coupled Wheat Germ Extract System for RNA isolation, gene expression and gel-shifts assays (EMSA) respectively. (1477)

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)

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)

Notes: Studies were performed in HepG2 cells. Cells were transfected with a hepatocyte nuclear factor-6 (HNF-6)–responsive promoter firefly luciferase construct (3µg), various forms of the HNF-6 protein in an expression vector (400ng) and a Renilla luciferase control vector (pRL-null Vector) driven by the liver pfk-2 promoter (not responsive to HNF-6; 500ng). The ability of the various HNF-6 isoforms to drive luciferase expression was normalized to Renilla luciferase activity, and the luciferase activity was measured with the Dual-Luciferase^® Reporter Assay System. The various HNF-6 constructs were also expressed in vitro with the TNT^® Coupled Wheat Germ Extract System and used for gel shift assays. (0839)

Notes: This paper describes a method for quantitation of luciferase mRNA by in vitro translation using Promega TNT^® Coupled Wheat Germ Extract System. (Rabbit reticulocyte lysate could not be used because of luciferase quenching problems). In this reporter gene assay, COS cells were transfected with a firefly luciferase reporter plasmid driven by promoters/enhancers of varying strengths and total cellular RNA was isolated and translated in vitro using the TNT^® System. To normalize expression, a CMV Renilla luciferase or beta-galactosidase vector was used as a control. To measure luciferase activity either Promega Luciferase Assay System or Dual-Luciferase^® Reporter Assay System was used. (2047)

Notes: Expression of luciferase was studied in the TNT^® Coupled Wheat Germ Extract System in the presence of circular and linear antisense (sulfide cross-linked) oligonucleotides that contained sequences complementary to the initiation codon region of firefly luciferase mRNA. Satisfactory antisense properties were displayed with modified circular oligonucleotides that contained a short random mismatching sequence. The application of these molecules to inhibition or preservation of enzymatic reactions may be useful for designing antigene technologies. (1486)

Notes: The authors used the Promega pSV-β-Galactosidase Control Vector, pGL3-Control and pGL3-Basic Vectors, Luciferase Assay Reagent, TNT^® Coupled Wheat Germ Extract Systems and TNT^® Coupled Reticulocyte Lysate Systems in their studies NF-κB. (0994)

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)

Notes: In this paper, a DNA fragment coding for the luciferase enzyme was used as a label in hybridization assays. Microwells were coated with anti-digoxigenin, digoxigenin-labeled capture probe was immobilized in the wells, and hybridization with the target DNA was allowed to proceed. Streptavidin-luciferase DNA complex was added to each well. Transcription/translation mixture (from the TNT^® T7 Coupled Wheat Germ Extract System) was added to the wells and incubated for 30 minutes. Luminescence was then measured using a liquid scintillation counter. (1341)

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: Trans (methionine plus cysteine) [35S]labeled proteins were synthesized using the TNT® Wheat Germ Extract System. Wild type and mutant (v-DeltaNLS and v-SPW) v-Rel proteins were tested for DNA binding in EMSAs. v-SPW cannot bind DNA but can form heterodimers with NF-kappaB p52. This v-SPW/ NF-kappaB p52 heterodimer can bind DNA in vitro. (1821)

Notes: The cDNA for type IV adenylyl cyclase (ACIV) was expressed in the TNT^® Coupled Reticulocyte Lysate System in the presence of [³⁵S]methionine. Adenylyl cyclase activity was assayed, and the protein was immunoprecipitated with antibodies to ACIV. The adenylyl cyclase was activated by bovine brain Gs and forskolin and was quite stable. This report showed that ACIV can be produced in vitro with the activity of baculovirus-produced ACIV. (1777)

Notes: The Vitamin D Receptor (VDR) and Retinoid X Receptor beta (RXR-beta) cDNAs were transcribed and translated in the TNT^® Coupled Wheat Germ Extract System. The proteins were incubated with and without calreticulin and probe in gel retardation assays. Calreticulin inhibits VDR homodimers and VDR/RXR heterodimers from binding VDRE DNA sequences. (1816)