Notes: A minimal promoter from the gene encoding BiP was cloned into the pGL3-Basic Vector. The resulting construct was transfected into HEK293 cells and, 48 hours post transfection, cell lysates were prepared using Glo Lysis Buffer. The Bright-Glo™ Luciferase Assay System was then used to assess levels of luciferase expression.  As a transfection control, cells were co-transfected with the pSV-β-Galactosidase Control Vector.  (3229)

Notes: CHO (Chinese hamster ovary) cells were plated at a density of 2 × 10⁴ cells per well in 24-well plates or at 8 × 10⁵ cells/flask in 80cm² culture flasks to prepare for transient transfection. After 24 hours, the cells were transfected with 250ng of DNA/well using FuGENE® 6 Transfection Reagent at a 2:1 ratio or 1µg DNA/flask using 20µl of FuGENE® 6 Transfection Reagent. After 5 hours, the transfected cells were treated and either incubated overnight for the 24-well plates or harvested for an IP assay for the flasks. (4404)

Notes: Having observed that blunt 27mers had increased potency in RNAi compared to 21mers or 27mers with 3' or 5' overhangs, these authors investigated what differences may account for these changes in gene silencing activity using the same target sequence in enhanced green fluorescent protein (EGFP). For one experiment, a PCR-generated fragment of the EGFP coding region spanning sites EGFPS1 and EGFPS2 was cloned into psiCHECK™-2 Vector in both sense and antisense orientations. Also, a PCR-generated fragment of the human heterogeneous nuclear ribonucleoprotein H (hnRNPH) coding region spanning sites H1 and H3 was similarly cloned in sense and antisense orientations. HEK293 cells were transfected with 150ng EGFP sense and antisense vectors plus EGFPS2 or control duplex RNAs. HCT116 cells were transfected with 100ng sense and antisense hnRNPH vectors with H3 or control duplex RNAs. The Dual-Luciferase^® assay was used to evaluate luciferase expression 24 hours post-transfection. In a separate EGFP RNAi experiment, the Steady-Glo^® Luciferase Assay System was used to monitor firefly luciferase activity to normalize transfection of HEK 293 cells. A further RNAi experiment targeted the firefly luciferase gene in the pGL3-Control Vector cotransfected with 20, 2 or 0.4 nM siRNA duplexes into HeLa cells. After 48 hours, the cells were lysed and 10µl tested using the Luciferase Assay System. To test the level of expression of human La antigen targeted for gene silencing, total RNA was harvested from HeLa cells using the SV 96 Total RNA Isolation System, reverse transcribed and used in real-time PCR. (3289)

Notes: Type 1 angiotensin II receptor-Renilla luciferase (AT1R-Rluc), and β-arrestin1 and 2 GFP  fusion constructs (βarr1-GFP and βarr2-GFP) were created for BRET protein interaction assays. Combinations of AT1R-Rluc and β-arrestin-GFP constructs were transfected into COS-7 cells. The COS-7 cell cultures were then activated with 100nM angiotensin II in the presence of 60μM EnduRen™ Live Cell Substrate, and BRET fluorescence readings were taken at 475 and 515 nm over a 1 hour period. The authors also describe analysis of helix I mutants of β-arrestin1 and β-arrestin2 in similar β-arrestin-GFP construct BRET studies.  Data are displayed as a ratio of fluorescence readings with both constructs compared to fluorescence from the AT1R-Rluc construct alone.  (3256)

Notes: The authors examine and characterize HGS-ETR1 (Mapatumumab), a fully human agonistic monoclonal antibody with high affinity and specificity for TRAIL-R1. HGS-ETR1 induced cell death in tumor cell lines was mediated by the activation of the extrinsic and intrinsic death signaling pathways. The tumor cell lines Colo205, HCT116, H460, H2122, ST486, SW480, RL95-2, WU.86.86, ES2, A498, WM793, SNU398, JURL-MKI and TTn were plated at 10,000 cells/well in 96-well white, opaque plates. The tumor cells were treated with the HGS-ETR1 or a isotope control antibody (IC_mAB). After 48 hours, cell viability was determined by the CellTiter-Glo® Luminescent Cell Viability Assay. Luminescence was detected using a Northstar luminescent plate reader. Caspase 3/7 activity was assessed in the HGS-ETR1 and ICmAB treated tumor cells using the Apo-ONE™ Homogeneous Caspase-3/7 Assay. The cells were plated at 10,000 cells/well into black-walled 96-well plates and treated with the HGS-ETR1 and IC_mAB for 6 hours at 37°C. Caspase activity was measured by reading the plates at 405nm using a fluorometric plate reader. (3314)

Notes: CEDIA assays (cloned enzyme donor immunoassays) utilize antibodies to specific analytes that can also bind one of the two beta-galactosidase fragments. The beta-galactosidase fragments can associate with each other provided that the fragment bound to the analyte is not immunoabsorbed, thus blocking the fragment interaction.  The Beta-Glo^® Assay System was compared to colorimetric and chemiluminescent detection systems for use in a CEDIA assay with valporic acid as analyte. The Beta-Glo^® Assay System gave the best performance in this assay system.  (3224)

Notes: HIV-1 integrase (HIV-IN) catalyzes a two-step reaction that results in HIV-1 provirus incorporation into the host cell genome. The steps involve an endonucleolytic 3’-processing (3P) followed by a strand transfer (ST) reaction. In past research it has been demonstrated that small molecule inhibitors of the in vitro activity of the 3P reaction only, lack antiviral activity, while inhibition of the HIV-IN ST reaction has been shown to be the key to effective suppression of viral replication in vivo. In the ST inhibitor realm, two series of diketo acids and naphthridine carboxamides have demonstrated antiviral activity in cell culture-based models. However, no compounds have completed clinical trials so far, due to either limited potency or high toxicity. Traditional integrase assays have been low-throughput, gel-based assays involving radiolabeled oligonucleotides. More recently high-throughput assays have been developed, but these microtiter-based assays, while amenable to automation, are complicated and labor intensive due to the need for plate coating and washing. The authors describe development of two robust, homogeneous time-resolved fluorescent energy transfer (TR-FRET)-based assays for HIV-IN 3P and ST reactions that are optimized for 384-well amd 1536-well plate formats. A screen for HIV-IN inhibitors was performed on a 1.36 x 10⁶ compound library, resulting in a series of novel HIV-IN inhibitors that preferentially block integrase ST activity and show potential for further development as new antiviral drugs. (3776)

Notes: The authors validated the DNA IQ™ System for manual and automated DNA purification from blood, tissue, bone, hair, epithelial cells and mixed stains such as semen. Automated DNA extraction was performed using the Beckman Coulter Biomek® 2000 workstation. Purified DNA (0.5–1.0ng) was then amplified using the PowerPlex® 16 BIO System and a 6% PAGE PLUS™ gel. Automated DNA purification and use of a single-amplification STR system greatly increased sample throughput. (3645)

Notes: Ba/F3 cells were transfected with constructs encoding either wildtype or mutant (Imatinib-resistant) Bcr-Abl kinase. Src-family kinase activity was assayed using the SignaTECT® Protein Tyrosine Kinase Peptide 2 and SAM® Biotin Capture Membranes. Cell viability was measured using the CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay. (3405)

Notes: Wildtype or p53-knockout mouse embryo fibroblasts (MEFs) were infected with 2 MOI A/Puerto Rico/8/34 (PR8) influenza virus. After washing, DMEM containing 1% FBS was added and the cells were grown for 8 or 24 hours before adding the Caspase-Glo® 3/7 Reagent. After 1 hour, luminescence was measured and the fold increase was determined by comparing the RLUs of infected with mock-infected cells. In addition, the Luciferase Assay System was used to assess the level of p53 induction after PR8 infection. Either human primary lung or A549 cells were transfected with a p53-responsive firefly luciferase vector, subsequently infected with 2 MOI PR8 and assessed for reporter enzyme activity 4–24 hours post-infection. (3318)

Notes: Bacterial cytolethal distending toxin (CDT) is known to induce apoptosis of immune cells, but little is known of the toxin from Campylobacter jejuni. In this study, the authors examined the effects of C. jejuni CDT on cultured monocytes. The human monocyte line 28SC was inoculated with 2µg Campylobacter membrane proteins per milliliter of culture. After 8 hours, the cells were harvested and assessed for caspase-8 or caspase-9 activity using the Caspase-Glo^® 8 and Caspase-Glo^® 9 Assays, respectively. The 28SC cells were pulsed with 6.2 µM camptothecin for 2 hours as a positive control for apoptosis. (3288)

Notes: To examine the methylation status of the DPYD promoter, genomic DNA was extracted from an RKO cell line and from peripheral blood mononuclear cells using the Wizard^® SV Genomic DNA Purification System. The isolated DNA was treated by sodium bisulfate modification followed by amplification and sequencing, or by DHPLC to determine CpG island methylation state. (3587)

Notes: The role of various Neisseria meningitidis serotype B [NMB lipooligosaccharides (LOS)] in TLR4-dependent cell activation was investigated. A known protein binding partner, myeloid differentiation protein 2 (MD-2), was expressed in a baculovirus system with a 6X-His tag and the insect medium incubated with purified hexa-, penta-, and tetra-acylated endotoxins metabolically labeled with [³H] or [¹⁴C] acetate. These complexes (containing 0.2–1ng of radiolabeled LOS) were then incubated for 1 hour at 25°C with 100µl of HisLink™ Protein Purification Resin. After the incubation, the resin was centrifuged, the supernatant removed and the resin washed 3–4 times in 500µl PBS containing 5mM imidazole before elution with 2% SDS. The presence of radiolabeled LOS was evaluated by liquid scintillation spectroscopy and results were expressed as the percentage of LOS captured. (3317)

Notes: The authors evaluated use of a dual-color reporter assay for plant gene expression using the red and green light-emitting click beetle luciferase genes from the Chroma-Luc™ vectors (pCBR-Basic Vector, pCBG99-Basic Vector and pCBG68-Basic Vector). Plant expression vectors were constructed based on the reporter plasmid, pBI221, which contains the CaMV35S promoter, GUS reporter and nos-terminator cassette. The GUS cassette was replaced by the CBRluc, CBF99luc and CBR68luc genes. Twenty-five microliters of the gold microcarrier (1.6 mm) coated with 2µg plasmid DNA and were used to bombard plant specimens. Six hours after bombardment, an aqueous solution of 0.1mM D-luciferin potassium salt was sprayed on the transiently-transfected plants and luminescence detected using a CCD camera system. For the color-specific detection, the researchers used interference filters for wavelengths greater than 610nm (high-pass filter) and wavelengths between 510nm and 560nm (band-pass filter). To test luciferase activity in cell extracts, cultured tobacco (BY-2) and onion epidermal cells were cotransfected by bombardment using the click beetle constructs and a plasmid with the 35S promoter driving Renilla luciferase. The cells were homogenized in Passive Lysis Buffer and 8µl of the prepared lysate was assayed using the Dual-Luciferase® Reporter Assay. A second plasmid containing the inducible CAB1 promoter from Arabidopsis thaliana, was made by replacing the firefly luciferase gene with the CBG99luc-coding sequence from the pCBG99-Basic Vector. Spinach leaves were transfected by bombardment with the CAB1 construct, exposed to different light conditions for 21 hours and luminescence levels detected by CCD camera and interference filters. In all cases, the authors were able to detect click beetle luciferase expression in plants and cultured cells. (3293)

Notes: This study investigated the role of the dual-specificity protein phosphatase, Viral Protein 2 (VP2) from chicken anemia virus in virus-induced immunosuppression. Mutations were introduced into the VP2 sequence by overlap-expression PCR. Serine/Threonine phosphatase activity of VP2 mutants was investigated using the Serine/Threonine Phosphatase Assay System. (3525)

Notes: SignaTECT^® Calcium/Calmodulin-Dependent Protein Kinase (CaMKII) Assay System was used to determine CaMKII activity in mouse hippocampus lysates. (3401)

Notes: The Caspase-Glo™ Assay was used to monitor apoptosis in bovine neutrophils. In these experiments, the researchers slightly modified the Caspase-Glo™ add-and-read procedure. One million neutorphils were treated with a combination of TNF-α and/or gliotoxin for 6 hours before being lysed in a solution of PBS supplemented with protease inhibitors and 1% saponin. The lysates were cleared, stored at -80°C, and then diluted before being analyzed with the Caspase-Glo™ Assay System. Data was presented as the relative light units for each sample. (3305)

Notes: A10 rat aortic smooth muscle cells were transfected with up to four plasmids using FuGENE® 6 transfection reagent with a reagent:DNA ratio of 3:1. The amounts of plasmid DNA were 25ng of a luciferase reporter construct, 5ng of the pRL-TK control vector, 25ng of myocardin expression plasmid, varying amounts of a plasmid encoding a constitutively active Notch intracellular domain or Hairy-related transcription-factor, and enough empty pcDNA3 vector to normalize total DNA concentration in each transfection. 48 hours post-transfection, cells were harvested, and luciferase activities were measured using a dual luciferase assay from Promega. (4276)

Notes: Through immunoprecipitation studies it was found that levels of the proto-oncogenic serine/threonine kinase Pim-1 are regulated by ubiquitination. The kinase was found to associate with the chaperone proteins Hsp70 and Hsp90. Hsp70 associated with the ubiquitinated form of Pim-1, and Hsp90 protected Pim-1 from degradation. To study the activity of Pim-1 in these associations with chaperone proteins, recombinant Pim-1 protein was mixed with a five-fold excess of recombinant Hsp70 or Hsp90, or left unbound in the presence of a binding buffer [10 mmol/L HEPES (pH 7.4), 100 mmol/L KCl, 5 mmol/L DTT, 20 mmol/L Na2MoO₄, 50 mmol/L ATP]. These complexes were then immunoprecipitated with a Pim-1 monoclonal antibody and washed four times in kinase buffer [25 mmol/L HEPES, 10 mmol/L MgCl₂, 0.5 μg/mL DTT]. Finally, 0.1μmol/L ATP and 400 μmol/L of peptide substrate were added and the reaction incubated for 5 minutes at 30° C. The Kinase-Glo^® Luminescent Kinase Assay was used to determine the activity of Pim-1 in its various bound states. It was found that all binding configurations of the kinase were active, underscoring the importance of efficient degradation of the kinase as a means for regulating its activity. (3264)

Notes: Three terminal repeat sequences (TR) from Kaposi's sarcoma-associated herpesvirus (KSHV) were PCR amplified and then purified using the Wizard^® PCR Preps DNA Purification System. The PCR products were 386, 424, and 307 base pairs in size.  These TR products were used in PARP1-DNA ELISA assays.  The authors also used the Wizard^® SV Genomic DNA Purification System to isolate DNA from a BC3 cell line infected with KSHV. The isolated DNA was used in real-time PCR to assess KSHV copy number in cells. (3232)

Notes: These authors studied the activity of oxygen-independent coproporphyrinogen III oxidase HemN, an enzyme involved in converting coproporphyrinogen III to protoporphyrinogen IX in heme and chlorophyll biosynthesis. The activity assay for HemN requires protoporphyrinogen IX oxidase to convert the end products of the HemN reaction to a detectable form. Recombinant protoporphyrinogen IX oxidase was expressed as a polyhistidine-tagged protein in E. coli strain BL21-Codon-Plus(DE3)-RIL and purified using the MagneHis™ Protein Purification System. (3569)

Notes: To generate mini-arrays, the Wizard^® SV 96 Plasmid DNA Purification System was used to isolate BAC DNAs from bacterial clones. The authors implemented the Wizard^® system using a Biomek 2000 Laboratory Automation workstation, and comment that the resulting DNA had low levels of contamination from the host E. coli, and was suitable for DNA amplification and subsequent array production. The performance of the mini-arrays was evaluated for gains and losses, with emphasis on reducing assay time and the amount of target DNA. They found that gains and losses of relatively large genomic regions such as full chromosomes and chromosomal arms could be reliably detected in <4 hours. (3542)

Notes: The authors studied the effects of Embryonic Stem Cell (ESC)-specific regulation on the Pou5f1 promoter in human and mouse cells. To examine the effect of knockdown of Oct4 and Sox2 (two genes involved in ESC regulation) on the Pou5f1 promoter, a 3kb fragment of the human POU5F1 promoter was cloned into pGL3-Basic Vector and 100ng cotransfected with 100ng shRNA plasmids into mouse E14 ESCs. Five nanograms of pRL-SV40 Vector served as a transfection control. For the enhancer assay, a 461bp fragment of genomic DNA containing the SRR2 enhancer of Sox2 was amplified and cloned into the pGL3-Promoter Vector. The same amounts of plasmid, shRNA and transfection control were transfected into E14 ESCs as in the Pou5f1 promoter assay. To investigate gene knockdown in 293T cells, 5ng of the two open reading frame (ORF) constructs (the Luc-Sox2 and the Luc-Pou5f1 ORFs cloned into the psiCHECK™-2 Vector) were cotransfected with 100ng shRNA plasmid. The outcome was examined 48–60 hours post-transfection using the Dual-Luciferase^® Reporter Assay System. (3291)

Notes: Mice that overexpress Klotho exhibit an extended lifespan and delayed aging. In this study, the authors show that Klotho protein protects against oxidative stress and activates the FoxO transcription factors, inducing expression of manganese superoxide dismutase. Two luciferase constructs were made, one with luciferase under the control of the Fas ligand promoter and one under the control of the human SOD2 promoter. HeLa cells were transfected with one of the two luciferase constructs and the pRL-CMV Renilla control vector. Transfected cells were treated with or without Klotho protein, and cell lysates were analyzed using the Dual-Luciferase^® Assay. Klotho protein stimulated the activity of the Fas ligand gene promoter and the SOD2 promoter. (3667)

Notes: These authors describe use of the Beta-Glo^® Assay System to analyze beta-galactosidase reporter enzyme concentrations from yeast three-hybrid experiments. The authors optimized the Beta-Glo^® assay with various Saccharomyces cerevisiae strains. The optimal incubation time using the Beta-Glo^® reagent on yeast was one hour, whether the cells were in stationary or exponential growth phase. Data is presented as arbitrary light units from the luminometer. (3295)