Notes: U2OS and H1299 cells were transfected with expression vectors and analyzed for either protein levels or mRNA levels. Cells for mRNA level detection were transfected with the ViaFect™ Transfection Reagent (details not provided). Total RNA from siRNA treated cells was isolated and used in semi-quantitative RT-PCR with the PCR portion provided by the GoTaq® Hot Start Green Master Mix. The proteins ARID3A and ARID3B were expressed in with the TNT® Coupled Reticulocyte Lysate System for use in electrophoretic mobility shift assays. (4677)

Notes: The authors of this paper studied the targeting mode of the Parkinson disease-associated kinase Pink1. They constructed a number of truncation and deletion mutants in the pGEM®-4Z Vector, and verified the identity of these clones using next-generation sequencing. The authors then expressed radiolabeled versions of the various Pink1 constructs using the TNT® Coupled Rabbit Reticulocyte Lysate System. These labeled proteins were used in mitochondrial localization studies.   (4563)

Notes: These authors characterized inflammatory caspase substrates using an enzymatic enrichment method for caspase 1,-4 and -5 cleaved proteins and mass-spectrometry. To confirm that caspase-1 cleaved the putative substrates, some substrates were expressed and fluorescently labeled using the TNT® T7 Transcription/Translation System and the FluorTect™ Green_Lys System. The proteins were then treated with recombinant caspase-1, and the progression of the reaction tracked via SDS-PAGE. The authors also used the CytoTox™ ONE Homogeneous Membrane Integrity Assay to track membrane permeabilization in relation to caspase cleavage and IL-1B release. (4252)

Notes: The authors were interested in examined the relationship between pregnane X receptor (PXR) and protein arginine methyltransferase 1 (PRMT1). Cells were transiently transfected for 60 hours including chemical treatment, and the Luciferase Assay System was used to analyze reporter activity. For the Checkmate™ Mammalian Two-Hybrid Assay, CV-1 cells were transfected with the pBIND Vector containing PXR, pACT Vector containing PRMT1 and pG5luc Vector. After 12 hours, the cells were treated with rifampicin and 48 hours later, luciferase activity was measured. Full-length PRMT1 protein was synthesized using the TNT^® SP6 Coupled Reticulocyte Lysate System and used in a GST pulldown assay. (4027)

Notes: The authors characterized a novel caspase 8-like activity that cleaves Bid and activates the mitochondrial apoptotic pathway. This activity was purified from rat liver lysosomal extracts and later identified as chymotrypsin B (CtrB). CtrB was previously thought to be expressed only in the pancreas, but the authors were able to detect crtB RNA in total RNA from primary rat hepatocytes and a rat hepatoma cell line (RH-35) using RT-PCR and the Access RT-PCR System. To confirm the intralysosomal localization of Crt B, the authors transfected RH-35 cells with an expression vector encoding CrtB tagged with green fluorescent protein. Prior to transfection, synthesis of functional protein from the expression vector was confirmed by in vitro transcription and translation using the TNT^® Coupled Transcription Translation System and [³⁵S] methionine. (3889)

Notes: The authors performed protein pull-down assays to characterize the interaction of ORAI1 and STIM1, two protein components of the calcium-release calcium current. His₆-STIM1 C terminus and ORAI1 were synthesized using the TNT® Coupled Reticulocyte Lysate System in the presence of ³⁵S, and His₆-STIM1 C terminus was immobilized using MagZ™ Binding Particles. An aliquot of the TNT® reaction expressing ORAI1 was added to the particles, and proteins were washed, eluted using increasing concentrations of imidazole (10–40mM) and analyzed by SDS-PAGE. In a second set of pull-down assays, His₆-STIM1 C terminus was used to pull down ORA1 N- and C-terminal fragments expressed as GST fusion proteins. The His₆-STIM1 C terminus protein was purified from transiently transfected HEK293 cells using the MagneHis™ Protein Purification System. (3781)

Notes: Nuclear transport of hypoxia-inducible factors (HIF) allows these factors to activate transcription of genes including epo,vegf an glut1 to maintain oxygen homeostais in cells. In this study the authors synthesized HIF-1α,β and HIF-2α in vitro, and used the expressed proteins in binding studies to determine the nature of HIF binding to nuclear pore complex proteins. The HIF proteins were transcribed and translated in vitro in the presence of ³⁵S-methionine using the TNT^® Coupled Reticulocyte Lysate System according to the protocol. After incubation, 10µl of the reaction batch was allowed to bind to the immobilized fusion-proteins importin alpha3, alpha5, and alpha7. The direct interaction of HIF-1α with alpha importins was dependent on functional nuclear localisation signal within the C-terminal region of the protein. In contrast, the supposed N-terminal NLS was not effective. In a typical experiment 100µl GST beads were pre-equilibrated in IP buffer (20mM Hepes pH 7.5, 100mM KOAC, 0.5mM EGTA, 5mM MgOAc, 250mM sucrose, 4°C), mixed with 15µg GST-fusion proteins and His-tagged importin beta and incubated at 4° C for 1 h. (3947)

Notes: This article examines the interaction between CAR, a member of the nuclear steroid/thyroid hormone receptor family, which translocates from the cytoplasm to the nucleus when cells are exposed to phenobaritol, and the membrane-associated subunit of protein phosphatase 1 (PPP1R16A, or abbreviated as R16A), which is a novel CAR-binding protein. The R16A protein, it was expressed using the TnT^® Coupled Reticulocyte Lysate System and labeled with ³⁵S. This protein was incubated with GST-hCAR-fusion protein attached to a glutathione resin; the resin was washed, and the bound protein was separated by PAGE and detected by autoradiography. Affinity-tagged R16A was expressed in HepG2 cells, purified and separated by SDS-PAGE. The two major bands were excised, digested with Sequencing Grade Modified Trypsin, lyophilized, resuspended in acetonitrile and subjected to mass spectrometric analyses. The CheckMate™ Mammalian Two-Hybrid System was used to examine the interaction between wildtype and mutated R16A. Forty-eight hours posttransfection into HepG2 cells or 16 hours after DNA injection into mice and liver homogenization, the luciferase activities were measured using the Dual-Luciferase^® Reporter Assay System. (3752)

Notes: These authors prepared a complete collection of Vibrio cholerae ORF clones using an automated amplification and cloning procedure. They then tested this set of clones for protein expression and capture using a nucleic acid programmable protein array method. To do this, they used the TNT^® T7 Coupled Reticulocyte Lysate System to perform in situ transcription/translation of cDNA clones containing a GST fusion tag. Proteins were captured using an anti-GST antibody and subjected to further analysis. (3879)

Notes: The authors developed an in vitro selection system that is based on ribosome display and favors identification of 5´-untranslated regions (UTRs) with high translation efficiencies. A 5´-UTR random library was created in which the 5´-UTRs were upstream of a polyhistidine-tag/Renilla luciferase-coding region. In vitro transcripts from this library were translated in vitro using the Flexi^® Rabbit Reticulocyte Lysate System. The authors preferentially selected mRNAs with high translational efficiencies by shortening the translation time and capturing ternary complexes of mRNA, ribosome and nascent proteins. These complexes were captured using MagneHis™ Ni Particles. RNA was extracted from these complexes and used as a template in RT-PCR for the next round of selection. Before and after each round of selection, 9µl of RNA was translated in vitro, and 20µl of translated product was removed every 5 minutes to measure Renilla luciferase activity and monitor translation efficiency. Renilla luciferase was measured using the Renilla Luciferase Assay System. After two rounds of selection, RT-PCR products were cloned into a pUC18 vector, the sequences of the resulting plasmids were confirmed, and 0.5µg of plasmid was translated in vitro using the TNT^® T7 Coupled Rabbit Reticulocyte Lysate System to further evaluate translation efficiency. (3963)

Notes: The authors show that SepN, a selenoprotein of unknown function, and ryanodine receptor (RyR) intracellular calcium release channel are both required for normal muscle development in zebrafish. Furthermore SepN and RyR interact, and SepN is required for full activity of the RyR channel. As part of their study, the authors expressed SepN as a polyhistidine-tagged (8X His) protein in TNT^® Coupled Reticulocyte Lysate System and purified it using the MagZ™ Protein Purification System. The TNT^® reaction was modified to optimize selenocysteine incorporation efficiency; the reaction contained 80% rabbit reticulocyte lysate (RRL), 1mM methionine, 0.4mM spermidine, 0.01µg/ml SepN-8X-His DNA and 300nM of the C-terminus of Secis Binding Protein 2 , which is required for efficient incorporation of selenocysteine in RRL-based reactions. (3898)

Notes: The authors examined the interaction between various proteins involved in mRNA deadenylation and degradation using GST pull-down assays. Cytoplasmic poly(A)-binding protein (PABPC1) was expressed in E. coli as a GST fusion and immobilized using the MagneGST™ Glutathione Particles. This bait protein was incubated with various [³⁵S]Methionine-labeled prey proteins expressed in the TNT^® Coupled Reticulocyte Lysate System. Bait:prey complexes were washed, eluted with 1X SDS loading buffer, then analyzed by SDS-PAGE to determine which proteins interacted. (3782)

Notes: To explore the interaction of liver receptor homolog (LRH)-1, a known suppressor of sterol regulatory element-binding protein (SREBP) transcriptional activity, human LRH-1 was reverse transcribed then amplified by PCR from total RNA from HepG2 cells. The amplification product was ligated into the pTargeT™ Mammalian Expression Vector to create pTarget-LRH1. For reporter experiments, a PCR fragment that encompassed the 1.3kb 5’-promoter region of the human small heterodimer partner (SHP) gene was cloned into the pGL3-Basic Vector (designated pSRB). The pGL3-Promoter Vector was used to construct pLRHREx3, which contains three LRH-1 response elements, and the insert was generated using synthetic oligonucleotides. HEK293 cells were cotransfected with 0.2µg of a promoter-firefly luciferase construct, 0.1µg of a SREBP expression plasmid, 10ng of phRL-TK Vector and 0.2 or 0.6µg of pTarget-LRH1. Alternatively, the cotransfected plasmids were 0.2µg of pSHP, 0.1µg of pTarget-LRH1, 10ng of phRL-TK Vector and 0.2 or 0.6µg of a SREBP expression plasmid. The pLRHREx3 construct (0.2µg) was cotransfected with 0.1µg of a LRH-1 expression plasmid, 0.2µg of pCMXPGC-1α (peroxisome proliferator activated receptor γ coactivator-1α), 10ng of phRL-TK Vector, and 0.1 or 0.3µg of a pSREBP expression vector in HEK 293 cells. Luciferase expression was assayed 48 hours post-transfection using the Dual-Luciferase^® Assay Reporter System. To express SREBPs and LRH-1 in vitro, inserts were ligated into the pTNT™ Vector, synthesized using the TNT^® Coupled Transcription/Translation System with radiolabeled methionine. Ten microliters of the ³⁵S-labelled protein was then used in a GST-pulldown assay. (3692)

Notes: The protein Lin-28 is a translational enhancer in differentiating myoblasts; one target of Lin-28 is insulin-like growth factor 2 (IGF-2). The authors showed that Lin-28 increased expression of an IGF-2 reporter construct in vitro. [³⁵S]-methionine-labeled, His-tagged Lin-28 was expressed in the TNT^® Coupled Reticulocyte Lysate System and purified using the MagZ™ Protein Purification System. Increasing amounts of purified Lin-28 protein were added to a TNT^® Coupled Reticulocyte Lysate System reaction containing an IGF-2 luciferase reporter vector, and as a result, luciferase expression was increased up to threefold. Experiments performed with an irrelevant His-tagged protein of equal size and with an equal number of methionine residues confirmed that the increase in luciferase activity was specific to Lin-28. Side-by-side experiments performed with a luciferase reporter vector without the IGF-2 regulatory element did not show increased luciferase activity. (3717)

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: 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: 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: 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: These authors characterized the interaction between whirlin, a protein component of stereocilia in the inner ear, and the membrane-associated guanylate kinase protein p55. Portions of p55 and whirlin were expressed in the TNT^® T7 Coupled Transcription/Translation System as ³⁵S-labeled protein. GST-fusion proteins of the guanylate kinase of p55, the PDZ3 domain of whirlin or GST alone were expressed in E. coli. Interaction between the ³⁵S-labeled proteins and the GST-fusion proteins were examined in GST pull-down assays using the MagneGST^® Pull-Down System. GST alone was used as a negative control. Briefly, the MagneGST^® particles were washed eight times with wash/binding buffer supplemented with 0.05% Nonidet^® P-40, and bound proteins were resuspended in 2X SDS sample buffer and analyzed on 12.5% SDS polyacrylamide gels. (3683)

Notes: To examine further the phenotype of a known tissue-nonspecific alkaline phosphatase (TNSALP) frameshift mutant, the cDNAs for wildtype TNSALP and TNSALP (1559delT) were subcloned into pALTER^®-MAX Vector. Three cysteine residues were replaced with serines using the Altered Sites^® II Mammalian Mutagenesis System. The substitution mutations were confirmed by DNA sequencing and the plasmids transfected into CHO cells. The size of the TNSALP (1559delT) product was compared to wildtype TNSALP using the TNT^® T7 Coupled Reticulocyte Lysate System and [³⁵S]methionine ⁄ cysteine with or without Canine Pancreatic Microsomal Membranes. The proteins were analyzed using SDS-PAGE and fluorography. (3519)

Notes: The GG2 element located upstream of the human insulin gene was mutated and cloned into a firefly luciferase construct. Two pancreatic mouse cell lines, ßTC-3 and Min6, were co-transfected with the various GG2 luciferase vectors using phRL-TK Vector as a normalization control. Luciferase expression was then assessed using the Dual-Luciferase^® Reporter Assay System. Three factors known to affect insulin gene expression were transcribed and translated using the TNT^® Coupled Reticulocyte Lysate System. The proteins were then used in a gel-shift assay with several DNA element oligos. A 38-40 kDa protein that bound to the GG2 element was identified.  This protein was isolated by DNA affinity chromatography, run on a SDS-PAGE gel and digested with 0.01µg/µl Sequencing Grade Modified Trypsin. Digestion products were then analyzed by Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and TOF/TOF tandem mass spectrometry. (3116)

Notes: Wildtype and Asp-multated SCL proteins were synthesized in vitro using coupled transcription and translation. (2720)

Notes: The TNT^® T7 Coupled Reticulocyte Lysate System was used to express precursors and truncated precursors of cowpea and Arabidopsis phosphoribosyl aminoimidazole synthetase (AIRS) in the presence of [³⁵S]methionine. Purified chloroplasts or mitochondria were added to the TNT^® T7 Coupled Reticulocyte Lysate in vitro transcription/translation reactions to monitor uptake of the protein into each organelle. The researchers also performed negative controls on the chloroplast or mitochondria uptake assays by adding 1 unit of apyrase or 20μm valinomycin, respectively.  (2688)