Notes: Specific neuronal connectivity is thought to be based on the expression of the protocadherins (Pcdh)--a family of membrane adhesion proteins. Each neuron expresses only a specific subset of the Pcdh genes. The authors of this paper used a bioinformatics approach to identify conserved, gene-specific regions upstream of the Pcdh genes. They showed that this specific sequence element (SSE) is involved in transcription regulation together with a conserved sequence element (CSE), and identified a potential interacting protein partner. To demonstrate promoter activity, the SSE-CSE region was cloned upstream of the luciferase gene in the pGL3 Basic Vector and its effect on luciferase expression was evaluated. The authors then isolated protein complexes that bound the SSE-CSE region and characterized the interacting proteins by mass spectrometry. The CCTC binding-factor (CTCF) was identified as a key molecule that binds and activates Pcdh promoters. As part of the study, human CTCF  and the CTCF-binding domain were expressed in the TNT® T7 Quick Coupled Transcription/Translation System. The in vitro expressed proteins were fluorescently labeled using the FluoroTect™ Green_Lys System and were used in EMSA to confirm interaction with the SSE-CSE region. (4249)

Notes: These authors describe use of a microfluidic device based on mechanically induced trapping of molecular interactions (k-MITOMI) for kinetic characterization of 768 biomolecular interactions in parallel. Using this approach, they measured the association and dissociation kinetics of numerous transcription factors with their associated consensus sequences. The TNT® T7 Quick Coupled Transcription/Translation System was used to express the transcription factors from linear templates, either on-chip or in bulk reactions. (4247)

Notes: These authors compared 6 different detection strategies for protein-protein interactions on protein arrays. They expressed HaloTag^® labeled bait proteins in a cell-free expression system, and captured these bait proteins onto coated glass slides using the HaloLink™ Array System. They then compared detection strategies using prey proteins labeled as follows: 1)³⁵S methionine, 2) fluorescence (BODIPY-FL) and 3) biotin labeling of lysine residues using modified Lys tRNA, 4) chemical labeling after expression, 5) HaloTag^® fusion, and 6) N-terminal FLAG tag. The authors evaluated signal:background ratios, adaptability to high-throughput screening, and ease of use. (3999)

Notes: The authors identified human glucokinase (hGK) as a substrate for the ubiquitin-conjugating enzyme system of rabbit reticulocyte lysate (RRL). Wildtype hGK, His₆-hGK and His₆-hGK mutants were expressed in the TNT^® T7 Quick Coupled Transcription/Translation System in the presence of [³⁵S]methionine and 10µM ubiquitin (in additional to the endogenous ubiquitin in RRL). Ubiquitinated and polyubiquitinated proteins were detected as size-shifted bands by SDS-PAGE and by two-dimensional polacylamide electrophoresis followed by immunoblotting with an anti-ubiquitin antibody. For some experiments, His₆-hGK and His₆-hGK mutants were isolated using the MagZ™ Protein Purification System prior to ubiquitination. (3718)

Notes: To explore the role of C/EBPb isoforms in regulating p53 expression during the cell cycle, the 1.7 kb murine p53 promoter was cloned into the pGL3-Basic Vector. Using TransFast™ Reagent, Swiss3T3 and 6629 (C/EBPb-null) cells were transfected with 0.1–0.75 µg of pGL3-1.7-kb p53 promoter construct with or without co-transfection of 0.25 µg of C/EBPb-2, and with 50 ng of pRL-TK Vector as an internal control. Twenty-four hours post-transfection, the cells were harvested and assayed for luciferase activity, normalizing reporter activity to Renilla luciferase activity. The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to either mutate or delete the –972/–953 cis-acting element carrying the C/EBPb-binding site within the p53 promoter, and 0.1–0.75 µg of the mutant constructs were then tested in the same reporter assay. The C/EBPb-1, -2, and -3 cDNAs were cloned into an expression vector and translated using 0.5µg of plasmid in the TNT^® T7 Quick Coupled transcription/translation system either in the presence of unlabeled or [³⁵S]methionine. The synthesized proteins were analyzed on 12% SDS-polyacrylamide gel. (3675)

Notes: The authors characterized a juvenile hormone response element in Drosophila melanogaster (DmJHRE1) and identified two proteins that bound to a DmJHRE1 affinity column. Proteins eluted from the column were digested with Sequencing Grade Modified Trypsin, subjected to liquid chromatography-tandem mass spectrometry and identified as FKBP39 and Chd64. DmJHRE1 transcription regulatory activity was confirmed using reporter constructs with DmJHRE1 sequences regulating expression of firefly luciferase in Drosophila L57 and S2 cells. A vector with Renilla luciferase and the Autographa californica multicapsid nucleopolyhedrovirus IE1 promoter was used for normalization. Luciferase activities were measured using the Dual-Luciferase^® Reporter Assay System. Potential interactions between FKBP39, Chd64 and several candidates proteins for the JH receptor were examined using the MagneGST™ Pull-Down System. Each bait protein was expressed as a GST-fusion protein in E. coli and immobilized using MagneGST™ Glutathione Particles. [³⁵S]Methionine-labeled prey proteins were expressed using the TNT^® T7 Quick Coupled Transcription/Translation System. (3784)

Notes: The authors confirmed the interaction of NKX3.1, a prostate-specific homeodomain protein, and topoisomerase I. To determine of this interaction was dependent upon nucleic acid, two types of pull-down assays were performed in the presence of DNase or RNase. For the GST pull-down assay, fragments of topoisomerase I were expressed as GST-fusion proteins, and NKX3.1 was expressed as an [³⁵S]methionine-labeled protein in the TNT^® Quick Coupled Transcription/Translation System. Equimolar amount of GST or GST-topoisomerase I, bound to glutathione sepharose beads, and NKK3.1 were precipitated . In addition, fragments of NKX3.1 were expressed as polyhistidine-tagged proteins and captured using the MagZ™ Binding Particles. Equal molar amounts of NKX3.1 and [³⁵S]methionine-labeled topoisomerase were incubated to examine protein interaction. (3716)

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: Sex-determining Y-box (SOX) 6 is a transcription factor downregulated in obesity-related insulin-resistant animals. The authors examined the interaction between SOX 6 and β-catenin, a protein that modulates cyclin D1 promoter activity. To characterize the physical interaction, in vitro binding assays were performed using GST-fused SOX 6 and deletion mutants of β-catenin, which were expressed as ³⁵S-labeled proteins in the TNT® T7 Quick Coupled Transcription/Translation System. The GST-fusion proteins were bound to MagneGST® particles and allowed to interact with the β-catenin mutants. Purified GST was used as a negative control to determine nonspecific protein binding. The authors were able to identify the protein domains necessary for SOX 6/β-catenin interaction. Similar binding assays were performed with GST-β-catenin and ³⁵S-labeled T-cell factor in the presence or absence of SOX 6 to show that SOX 6 does not interfere with the binding of β-catenin to TCF. (3685)

Notes: The authors of this study determined the membrane topology of reticulon 3 (RTN3), an integral membrane protein that is expressed at high levels in neruons and has been show to negatively regulate the activity of BACE1 (Beta site APP-Cleaving Enzyme). Disruption of RTN3 is associated with incidence of dystrophic neurites in AD brain. RTN3 was translated using the TNT^® Quick Coupled Transcription/Translation System in the presence of Canine Microsomal Membranes and labeled using the Transcend™ Non-Radioactive Translation Detection System.
(3860)

Notes: Previously, the POU domain of Brn-3a was shown to interact with p53 and increase cell survival. In this article, the authors explored the possibility that Brn-3b, which shares a POU domain 95% identical to Brn-3a, may interact with p53 and affect its role in apoptosis. To test the protein:protein interaction, GST-Brn-3b fusion protein was bound to glutathione Sepharose beads and incubated with ³⁵S-methionine labeled full-length or truncated p53, prepared using the TNT^® T7 rabbit reticulocyte lysate. The luciferase control included in the kit was used as the noninteracting protein control. After washing, the bound proteins were resolved by 12% SDS-PAGE, and the bands examined by radiography. To examine the effect of Brn-3b on two p53-regulated genes, Bax and p21^cip1/waf1, ND7 cells were transiently transfected with empty vector, Brn-3b, Brn-3a or p53, or Brn-3 with p53. The reporter gene cotransfected was under the control of wildtype Bax, wildtype p21^cip1/waf1, or mutant Bax. A control vector (Renilla luciferase driven by the thymidine kinase promoter) was used for normalization. Forty-eight hours posttransfection, the cells were harvested and reporter levels assessed using the Dual-Luciferase^® Reporter Assay System. (3597)

Notes: To better understand the specificity and function of the 12 amino acid PAM2 (PABP-interacting motif 2) domain from the HECT E3 ubiquitin ligase (HYD), possible binding partners were identified. One set of potential partners, the anti-proliferative Tob proteins, were radiolabeled with ³⁵S and expressed in vitro using a TNT^® Quick Coupled Transcription/Translation reaction. The PAM2 domain from HYD and poly(A) binding protein were fused with a GST domain, expressed and purified from E. coli. Using the MagneGST™ Pull-Down System, the interaction between the PAM2 domain and the Tob proteins was confirmed by autoradiography. (3400)

Notes: Human arsenic methyltransferase (AS3MT) was cloned and mutated to produce allozymes for further analysis. COS-1 cells were transfected with constructs encoding the wildtype enzyme and the synthetic allozymes using TransFast™ Transfection Reagent. The wildtype enzyme and allozymes were transcribed and translated using TNT^® T7 Coupled Reticulocyte Lysate System in the presence of RNasin^® Ribonuclease Inhibitor. (3383)

Notes: To create a short hairpin RNA (shRNA) to Mcl-1, sense and antisense oligos were annealed and ligated into the psiSTRIKE™ Neomycin Vector. HeLa cells were then transfected with 5µg of linearized Mcl-1 shRNA plasmid using electroporation, and Geneticin^® was used to select for stable expression. The level of Mcl-1 was determined by immunoblotting. These cells, along with wildtype HeLa and stable control vector cells, were assessed for the level of mitochondrial apoptogenic factor release by isolating the mitochondria and exposing them to polyhistidine-tagged Bim, an apoptotic cascade protein. After a 30-minute incubation, the mitochondrial supernatant was analyzed for cytochrome c, Smac and HtrA2 in a Western blot assay. Also in this study, wildtype and mutated Mcl-1 proteins were expressed in vitro using 1µg of plasmid DNA with the TNT^® T7 Quick Coupled Transcription/Translation System and labeled with ³⁵S-methionine. One microliter of each reaction was then used in a 20µl caspase cleavage reaction with 5–100nM caspase-3 or -8, incubated for 20 minutes and analyzed by SDS-PAGE. (3389)

Notes: To test the effect that Xenopus laevis UDP-glucose dehydrogenase (xUGDH) expression has in mammalian cells, the xUGDH ORF was amplified, purified, A-tailed and cloned into the pTARGET™ Mammalian Expression Vector. Two clones were selected: one in the sense orientation and one in the antisense orientation. The constructs were confirmed by DNA sequencing and expression in a TNT^® T7 in vitro transcription/translation system. Five micrograms of the plasmids were transfected into AoSMCs and human aortic smooth muscle cells, and UGDH activities werw measured 48 hours post-transfection. (3497)

Notes: To examine the putative role that SUN1, a nuclear envelope (NE) protein with a C-terminal SUN (Sad1/UNC-84 homology) domain, may play in the inner nuclear membrane, the murine SUN1 cDNA was subcloned into the pCI-neo Mammalian Expression Vector. Primers containing the myc or HA tag sequence were used to amplify the pCI-neo construct, adding tags both 5’ and 3’ of the SUN1 cDNA. Emerin, a marker for the NE lumen, was also subcloned into the pCI-neo Vector and a 3’ myc tag added. These constructs were translated and labeled with ³⁵S in vitro using the TNT^® T7 Quick Coupled Transcription/Translation System, and the radiolabeled proteins were used in a GST pull-down assay. In addition, NIH/3T3 cells were transiently transfected with myc-SUN1 constructs, fixed in methanol and then co-stained with anti-myc and anti-lamin A/C antibodies. (3511)

Notes: The authors of this study investigate the relationship between BRCA1 and activity of the progesterone receptor (PR) in mammary tumor cells. BRCA1 mutations confer increased risk for steroid hormone-responsive cancers such as endometrial and cervical cancers and prostate cancer. In this study, evidence for interaction between PR and BRCA1 is presented. Glutathione-S-transferase (GST) capture assays were used to determine if PR and BRCA1 interact directly. GST-PR fusion proteins are used to pull down in vitro transcribed and translated BRCA1. In vitro transcription and translation reactions were carried out using a TNT^® Rabbit Reticulocyte Lysate system. Interaction between BRCA1 and PR isoforms A and B was observed, and this interaction did not appear to require the presence of progesterone. The authors also showed that BRCA1 regulates expression of several progesterone-responsive genes. (3602)

Notes: In this study, researchers examined the role of tumour necrosis factor (TNF) receptor-associated factors (TRAFs) in signaling by the KSHV viral FADD-like interleukin-1-β-converting enzyme (FLICE)/caspase-8-inhibitory protein (vFLIP). The TRAF domain of TRAF2 was expressed in vitro from a plasmid construct using the TNT^® Quick Coupled Transcription/Translation System and labeled with [³⁵S]methionine. vFLIP was cloned in-frame with a carboxy-terminal GST tag, and the recombinant vFLIP–GST fusion protein was expressed in E. coli and purified using the MagneGST™ Protein Purification System. GST protein only was similarly prepared as a control. The vFLIP–GST fusion and control proteins were incubated with the radiolabeled recombinant TRAF2, and protein complexes were collected by GST pull-down, washed thoroughly and subjected to SDS–polyacrylamide gel electrophoresis. In vitro-translated proteins were visualized by autoradiography. (3327)

Notes: The authors investigated common variants of the APOA5 gene that have been associated with differences in plasma triglyceride (TG) levels. PCR fragments containing either the –1131T --> C promoter variant or containing both the –1131T --> C and –3G --> A promoter variants were cloned into the pGEM^®-T Vector System. The fragments were subsequently cloned into the pGL3 Basic Vector and transiently transfected into Huh7 and HepG2 cells along with the luciferase control vector, pRL-TK. The cells were lysed 48 hours after transfection and Luciferase activity was measured with the Dual-Luciferase^® Reporter Assay System. The function of the 1891T --> C variant in the 3´ UTR was tested the same way; with the exception that site-directed mutagenesis was performed to introduce the T --> C at position 1891 before the fragment was cloned into the pGL3 Basic Vector. The functionality of the Kozak sequence –3A --> G variant was determined by cloning cDNAs into the pGEM^®-7Zf Vector. Transcription/translation experiments were performed using the TNT^® Quick Coupled Transcription/Translation System and the proteins were labeled using the FluorTect™ Green_Lys System. In addition, a primer extension inhibition assay was performed using capped mRNAs generated with the Riboprobe^® System –T7 and the Ribo m⁷G Cap Analog. Ribosome binding reactions were performed using the Rabbit Reticulocyte Lysate System, Nuclease Treated. (3460)

Notes: To prepare expression-ready cDNA clones of 1589 putative full-length ORFs (from human and mouse genes) with an average size of 2.8 kb, a linear trap vector was created. Generated by PCR, this linear plasmid contained gene-specific sequences to allow homologous recombination. In addition, 5–10µg of a plasmid containing the same gene-specific sequence and the linear trap vector were purified using the Wizard^® SV 96 PCR Clean-Up System. The purified DNA was resuspended in water and transformed into E. coli cells. The plasmid purified after the recombination was transferred to a Gateway expression vector and 100–200ng expressed in the TNT^® T7 Quick Coupled Reticulocyte Lysate System with 0.2µl of FluoroTect™ Green_Lys tRNA. The proteins expressed were resolved using SDS-PAGE. (3439)

Notes: This study investigated the role of c-Maf in transcriptional regulation of IL-10 in macrophages. c-Maf cDNA was cloned into the pTNT™ Vector, and recombinant human c-Maf synthesized from that plasmid using the TNT^® T7 Quick Coupled Transcription/Translation System. (3347)

Notes: The authors performed a yeast two-hybrid screen using big mitogen-activated kinase 1 (BMK1/ERK5) as the bait and identified the scaffolding protein 14-3-3beta. To confirm this interaction, the cloned mouse BMK1 gene was expressed in the TNT^® T7 Quick Coupled Transcription/Translation System. The expressed protein was labeled with Transcend™ tRNA.  Using a GST-14-3-3beta fusion protein, a pull-down assay was performed and the direct binding confirmed after immunoblotting and staining with streptavidin-horseradish peroxidase (HRP).  The interaction of various BMK1 mutants were tested in a mammalian two-hybrid system and measured by the Dual-Luciferase^® Reporter Assay System. (3078)

Notes: The authors cloned and sequenced the dsRNA from Penicillium chrysogenum virus (PcV) and analyzed the gene products of the segmented virus. After reverse transcription followed by PCR to confirm the 5’ and 3’ ends of the dsRNA segments, RT-PCR was used to amplify the complete cDNAs for the four viral segments. The products were cloned into pGEM®-T Vector after A-tailing. To confirm the ORFs predicted for each of the four segments, the cDNAs were added to TNT® T7 Quick Coupled Transcription/Translation System reactions that included radiolabeled methionine. The resulting proteins ranged in size from 94-128 kDa and were separated on an 8% SDS-PAGE gel and analyzed by autoradiography. Gradient-purified PcV virions were digested with Sequencing Grade Modified Trypsin at 37°C for 18 hours and the digestion products were separated by reverse-phase HPLC on a C18 column. Two highly resolved peptides were selected for amino acid sequencing by automated Edman degradation. (3089)

Notes: This study showed that membrane type-1 matrix metalloproteinase (MT1-MMP) binds C1q, the recognition unit of complement C1, which activates the classical pathway of complement. The peptides involved in C1q binding were identified and found to be distinct from those involved in proteolysis. cDNA fragments encoding various peptide sequences were cloned into the pTNT™ Vector prior to expression in the TNT^® T7 Quick Coupled Transcription Translation System. Translation reaction products were used in a C1q binding assay. (3348)

Notes: In this study, the authors investigated the interaction between the orphan receptors Dax-1 and Ad4bp/SF-1. Specific interaction was detected when the two proteins were used in both yeast and mammalian two-hybrid systems. To further characterize this interaction, an in vitro pull-down assay was performed. Dax-1 protein or a dax-1 mutant lacking an LXXLL motif were transcribed and translated in vitro using the TNT^® T7 Quick System and Fluorotect™ Green_Lys BODIPY labeled tRNA.  The fluorescently labeled protein interacted with a Ad4BP/SF-1-maltose binding protein (MBP) fusion protein immobilized to an amylose resin.  After elution and SDS-PAGE, the fluorescently labeled protein was detected using a Hitachi FMBIO^® II Fluorescence Imaging System. The interaction between Dax-1 and Ad4BP/SF-1 was dependent on the LXXLL motif. (3237)