Notes: The authors describe the advantages of the Kinase-Glo™ kit for high throughput screening. Cyclin-dependent kinase 4 (Cdk4) was used as a model kinase to draw comparisons between the Kinase-Glo™ assay and a “gold standard” radioactive filter assay in terms of reproducibility and use in compound inhibition studies. There is excellent description of the buffers, equipment and conditions used in the study. (3058)

Notes: These authors demonstrated that the human CD1D gene has distal and proximal TATA boxless promoter sequences. Distal and proximal promoters to CD1D were cloned into the pGL3-Basic Vector to create reporter constructs. One construct contained the entire 4,986 base pair region containing distal and proximal CD1D promoter.  Transient transfections were performed using 5 x 10⁵ Jurkat cells in 24-well plates, 0.8 μg of pGL3 Basic Vector with the insert of interest, and 30ng of pRL-CMV Vector as a transfection normalization control. The Dual-Glo™ Luciferase Assay System was used to assay luciferase activities.  (3061)

Notes: Human polynucleotide phosphorylase (hPNPase^old-35) was originally identified as a gene that is upregulated during cellular differentiation and senescence. The authors of this study show that overexpression of hPNPase^old-35 results in the increased production reactive oxygen species (ROS) and the activation of the NF-κB pathway, resulting in expression of the inflammatory cytokines IL-6 and IL-8. HeLa cells were transfected with either empty pGL3-Basic or 3kB-Luc (pGL3-Basic containing three tandem NF-κB binding sites). Transfected cells were infected with an empty adenoviral vector or one containing hPNPase^old-35. Luciferase assays were conducted using the Luciferase Assay System, and signal was normalized to a pSV-βgal control. A 10- to 12-fold increase in luciferase activity was observed in the cells infected with the adenovirus containing hPNPase^old-35 compared to the control cells. This activation was inhibited by compounds that reduced ROS. The authors suggest that hPNPase^old-35 plays an important role in producing age-related inflammation. (3643)

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: The authors describe use of the CellTiter-Glo^® Luminescent Cell Viability Assay to assess viability of rat primary cortical neurons after treatment with Aβ42 peptide or Aβ42 peptide-BV2 murine microglial cell conditioned media. The CellTiter-Glo^® Luminescent Cell Viability Assay was also used to assess cell viability during Aβ42 peptide treatment of BV2 cells transfected with siRNAs targeting sequences in cathepsin B, cathepsin L, TIMP2, and AIF1 genes. (2842)

Notes: The LY294002 phosphatidylinositol 3-kinase (PI3K) inhibitor was used to identify Nonsteroidal Anti-inflammatory Drug-activated Gene (NAG-1) as a novel downstream target of the PI3K pathway during cell activation. For these experiments, HCT-116 cells were treated with 50μM LY294002 and NAG-1 protein expression was assessed by Western blotting. Gene upregulation during LY294002 treatment was measured with a luciferase reporter construct containing the NAG-1 promoter, the pRL-null Vector as a transfection control, and the Dual-Luciferase^® Reporter Assay System. (3262)

Notes: The antiproliferative drug C-1305 was added at 0.1-2 µM or amsacrine at 0.1-1 µM for 24 hours, and then the cells were cultivated for an additional 48 hours in a drug-free medium. Cell proliferation was determined using the CellTiter-Glo® Luminescent Cell Viability Assay. (3140)

Notes: The CytoTox 96^® Non-Radioactive Cytotoxicity Assay was used to determine the cytotoxic effect of lipophilic transfection reagents commonly used for siRNA transfection. Data are presented as the percent cell death observed in HeLa and IGROV-1 cells at 24 hours post-transfection. The researchers tested the effect of each reagent with and without siRNA during transfections.  (3178)

Notes: In this paper,  lipopolysaccharide (LPS) induced PKC activity in human monocytic cell (U937) extracts was measured with the Kinase-Glo^® Luminescent Kinase Assay.  1 x 10⁷ U937 cells were cultured in the presence or absence of 5 μg/ml LPS for 4 hours.  The cells were then washed in PBS and resuspended in a kinase/extraction buffer (40 mM Tris [pH 7.5], 20 mM MgCl₂, 0.1 mg/ml BSA) before sonication. These extracts were then diluted in a PKC reaction buffer (20 mM Tris [pH 7.5], 10 mM MgCl₂, 0.1 mg/ml BSA, 250μM EGTA, 400μM CaCl₂, 0.32mg/ml phosphatidylserine, 0.032mg/ml diacylglycerol) with 10μM ATP and 100μM neurogranin_(28–43).  Results were displayed as relative light units compared to a control.  The researchers also mention normalizing the results by the Bradford protein determination assay. (3069)

Notes: Researchers cloned the Photinus and Renilla luciferase ORFs into the pSP64 Poly(A) Vector to create a dual-reporter vector named SP6P. A similar vector, SP6R.4G(-508/-3).P, was created in which a 5´ untranslated region from the Saccharomyces cerevisiae TIF4631 gene was cloned between the two reporter genes. These two vectors were used to transform yeast strains. The resultant transformants were lysed using Passive Lysis Buffer and a modified lysis procedure.   Lysates were analyzed for luciferase activities using the Dual-Luciferase^® Reporter Assay System and a TD20/20 luminometer. The researchers also cloned and sequenced the 5´  untranslated region of TIF4631 by using a RACE-PCR technique followed by cloning the PCR amplimers into the pGEM^®-T Vector. (2845)

Notes: In this article, a 2.5- and a 1.7-kb fragment of the myostatin promoter were amplified by PCR and cloned into the pGEM®-T Easy vector.  These clones were then subcloned into the pGL3-Basic Vector.  Deletion mutants of the constructs were made and used to transient transfect mouse muscle C₂C₁₂ cells.  The Luciferase Assay System was then used to analyze transfectants.  The researchers also injected luciferase-reporter constructs into mouse muscle.  Luciferase activity in the mouse muscle was examined by grinding isolated muscles in liquid nitrogen and resuspending them in Cell Culture Lysis Buffer.  Ten micron cryosections of the muscles were also used in immunohistochmical staining experiments.  For these experiments, the researchers used a 1:50 dilution of Promega’s polyclonal anti-luciferase antibody, a secondary antibody and tertiary fluorescein-labeled conjugate.  The slides were counter stained with DAPI. (3147)

Notes: The T7 RiboMAX™ Express RNAi System was used to create shRNAs to mouse and human neuron-restrictive silencer factor (NRSF). A scrambled sequence shRNA was also created with the system and used as a control. One to three micrograms of each shRNA were in transfecting NS20Y and HeLa cells.  The siRNA Target Designer (http://www.promega.com/siRNADesigner/) was used to design an siRNA against NRSF. (3208)

Notes: The authors studied the structure and function of Protein Kinase A (PKA) as a model for protein kinases. Two amino acids with contacts to other side chains, Thr197 and Ser338, were replaced with Alanine, expressed and purified from E. coli, and the enzymatic activity compared to that of wildtype PKA. The Kinase-Glo^® Luminescent Kinase Assay and the Kemptide peptide were used to determine the activities of the wildtype and mutant PKAs in the presence and absence of the known inhibitor, H7. (3290)

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: The Caspase-Glo^® 3/7, -8 and -9 Assays were used to  measure Caspase-3, -8, and -9 activities in mouse liver homogenates. The authors describe a modified procedure for making liver homogenates in a hypotonic extraction buffer.  Homogenates were cleared with a 15 minute spin at 13,000 x g and normalized to a 1mg/ml concentration before use the assays. Data comparing control and cSN50 peptide-treated animals at various time points are presented.  (3213)

Notes: COS-7 cells were harvested and total RNA isolated 24 hours after transfection with plasmids expressing trans-activating proteins. Five micrograms of RNA was reverse transcribed with oligo(dT) primer using the M-MLV Reverse transcriptase. One tenth of the reaction volume (2μl) was then used as template in PCR to amplify a gene presumed to be controlled by the trans-activators (pS2; 210bp) or a control gene (36B4 ribosomal phosphoprotein; 408bp). The PCR products were amplified using GoTaq^® DNA polymerase. (3203)

Notes: HeLa and Saos-2 were transiently transfected with luciferase reporter vectors made from the pGL3-Promoter vector containing various intron sequences from the human integrinα3 gene ITGA3. The first intron sequence was originally cloned by PCR cloning into the pGEM®-T Easy vector. Cell cultures were assessed for luciferase activity by making lysates with the Glo Lysis Buffer and assaying with the Steady-Glo® Assay kit. (3225)

Notes: After seeding 1 x 10⁵ cells/well in 96-wellplates, HeLa cells were allowed to attach for 24 hours. Cells were then treated with either media or WY-14643 (5-100 µM) in media for an hour before stimulation with 1 mM H₂O₂ (final concentration). Twelve hours later, hepatocyte injury was analyzed using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. (3167)

Notes: The authors cloned five distinct silencing suppressor proteins from five different plant viruses in order to examine the pathways involving both small interfering RNA and micro RNA in Arabidopsis thaliana. These viral factors [P1- HcPro of Turnip mosaic virus (TuMV), P38 protein of Turnip crinkle virus (TCV), P19 protein of Tomato bushy stunt virus(TBSV), P25 protein of Potato virus X, and the P15 protein of Peanut clump virus (PCV)] were inserted into a mammalian expression vector and tested for protein production using the TNT^® Quick Coupled Transcription/Translation System. The suppression effects of these plant viral proteins were also tested in HeLa cells. The CMV promoter was cloned from pRL-CMV into the pGL3-Basic Vector and both plasmids were transfected at 500ng each plus 1µg of each of the five suppressor-expressing vectors. After one day, 300ng siRNA targeting the firefly luciferase gene was added. Twenty-four hours later, the Dual Luciferase® Reporter Assay System was used to determine the ratio of firefly:Renilla luciferase expression and see if the viral suppressor protein had an effect. (3085)

Notes: Subconfluent ML20 cells were stripped of estrogens over three days then plated in a 96-well plate at 5000 cells/well. After overnight attachment, the cells were treated with fresh media containing 5% FBS plus ICI 182780 with or without each of the following: heregulin, epidermal growth factor; fibroblast growth factor 1 and β-estradiol or each of the factors individually. Under these various media conditions, the cells were treated with U0126. After five days, the cells were assayed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3154)

Notes: The authors studied the effect of nitric oxide stress on rat Islets of Langerhans cells and rat insulinoma cell line (RINm5F) and to determine if cross-linked hemoglobin (Hb-C)could mediate the stress and subsequent apoptosis.  The rat cells were treated with a nitric oxide donor SNAP and the DeadEnd™ Colorimetric TUNEL System was used to measure the effect of NO with or without Hb-C.  In addition, the cell viability was assessed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay.  Twenty-four hours after SNAP treatment, the level of NO₂ (a by-product of NO) was measured using Griess Reagent to determine the level of nitric oxide production induced. (3053)

Notes: Researchers used Promega’s cAMP-Dependent Protein Kinase Peptide Inhibitor to demonstrate that BAD kinase is phosphorylated through a cAMP-Dependent Protein Kinase (PKA) dependent pathway in Burkitt’s lymphoma BL-41 cells.  The PepTag® Non-Radioactive cAMP-Dependent Protein Kinase Assay was used to demonstrate PKA activity in BL-41 cell lysates.  In these experiments the researchers added cAMP with and without Promega’s cAMP-Dependent Protein Kinase Peptide Inhibitor (20μM) or Rp-cAMPS (100μM) to 0.2-2ug of BL-41 cell lysate.  Images of gels were depicted in the paper. (3134)

Notes: The authors examine the role of brain-derived neurotrophic factor (BDNF) in alcohol addition. RNA was isolated from primary rat hippocampal neurons cultured in the absence or presence of ethanol. The RNA was reverse transcribed using the Reverse Transcription System, and BDNF and GPDH RNAs were quantitated by fluorescent real-time PCR. BDNF and nerve growth factor (NGF) protein levels were monitored using the BNDF and NGF Emax^® ImmunoAssay Systems. (3441)

Notes: The authors used Xenopus laevis oocytes to show that unliganded thyroid hormone receptor (TR) recruits N-CoR (nuclear receptor corepressor) to modulate metamorphosis.  To study this influence, the cytoplasm of stage VI oocytes from X. laevis was injected with the indicated mRNAs [TR, retinoic acid receptor (RXR) and FLAG-tagged N-CoR].  The reporter plasmid TRE-Luc (0.33 ng/oocyte; thyroid hormone response elements from a Xenopus promoter driving expression of the firefly luciferase gene) and the control vector phRG-TK (0.03 ng/oocyte) were co-injected into the germinal vesicle (nucleus) after mRNA injection. After overnight incubation at 18°C, oocyte lysates were prepared by lysing six oocytes in 90µL 1X Passive Lysis Buffer. The Dual-Luciferase^® Reporter Assay System was then used to assay 7µl of the lysate. The researchers also used an expression vector (based on the pGEM^®-4Z Vector) containing the 5’ and 3’ untranslated regions of the X. laevis beta-globin gene flanking the multiple cloning site. (3088)

Notes: Cells were plated in a 96-well plate and incubated until near confluence. The cells were subsequently dried by removing the media before being placed in a humid atmosphere for 2-24 hours. Then the cells were rehydrated by incubating in media for two hours before being assayed for cell viability using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3163)