Notes: The authors developed a high-density protein array using a recombinant peptide library to map the epitope recognized by a commercially available anti-vitamin D receptor (VDR) monoclonal antibody. By screening 2304 overlapping VDR peptides, they were able to identify the 37-amino-acid epitope. The library was created by amplifying the 1.2kb VDR coding region, cleaning the PCR product with the Wizard^® SV Gel and PCR Clean-Up System, sonicating the PCR product, then cloning the VDR fragments into a bacterial expression vector that confers a glutathione-S-transferase (GST) tag. The epitope was verified by showing that the 37-amino-acid sequence was recognized in Western blot analysis and enzyme-linked immunosorbent assay (ELISA); the full-length VDR, also expressed as GST fusion protein, was used as a positive control. These GST fusion proteins were purified using the MagneGST™ Protein Purification System. (4154)

Notes: In this paper, FuGENE® HD reagent was used to transiently transfect HEK293 cells. Cells were plated on 24-well plates at 5 x 10e4 cells/well prior to transfection with 0.3µg plasmid DNA.

Notes: The authors of this paper demonstrate the utility of the HaloTag^® protein purification system for purifying functional proteins from mammalian cells. To this end five kinases were cloned into HaloTag^® vectors, expressed in and purified from HEK293T cells. To demonstrate functionality of the purified recombinant kinases, activity was measured using the appropriate ADP-Glo™ Assay Kinase Enzyme Systems. (4145)

Notes: Dysregulation of the NF-kB pathway has been associated with the formation of a wide variety of tumors and other cancers, as well as diseases, including chronic inflammation and immunodeficiency. Because of the association of constitutive NF-kB regulation and tumors, inhibition of the NF-kB pathway by small molecule antagonists was thought to be a means of reversing or halting the growth and spread of tumors. The authors screened compounds from a database (the NCGC Pharmaceutical Collection or NPC) of small molecule compounds: 52% of the compounds have been approved for human or animal use by the FDA, 22% were drugs approved for use in Europe, and another 25% either drugs approved in other countries or compounds that have been tested in clinical trials. The database served as a source from which to rapidly and efficiently identify already approved drugs that inhibited NF-kB. They used a quantitative high-throughput screening format. To identify small molecules that could inhibit the NF-kB pathway, the compounds were initially screened using a cell-based NF-kB lactamase reporter gene assay, with TNFalpha and MG132 as positive controls. (TNFalpha induced NF-kB coupled beta-lactamase activity, while MG132 blocked TNFalpha induction NF-kB-coupled beta-lactamase activity.) After several rounds of screening, 20 compounds were further studied for their NF-kB inhibition, with NF-kB luc2P HEK293 cells. After a concordance rate of 95% between the luciferase and beta-lactamase tests, compounds were additionally examined for their ability to affect caspase 3/7 activity, for the ability to disrupt the electrochemical gradient across the mitochondrial membrane in relation to cellular apoptosis, as well as tests of the inhibitors on cancer cell viability and affects on LDH release, an indicator of cell necrosis.
(4049)

Notes: pGL4.15[luc2P /Hygro] or pGL4.14 [luc2/Hygro] were used. (4265)

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: To help understand the molecular mechanism behind the biogenesis and assembly of photosystem II (PSII), the authors characterized the high chlorophyll fluorescence low psii accumulation19 (lpa19) mutation, which results in defective PSII biogenesis. To examine Lpa19 expression patterns in lpa19 mutant plants, the authors performed Western blot analysis using an anti-Lpa19 antibody raised against purified recombinant His-tagged LPA19 protein. This antigen was purified using the HisLink™ Resin. (4101)

Notes: In this study the luminescence-based BacTiter-Glo System and the GloMax 20/20 luminometer were used to detect ATP concentrations as low as 0.0001 nM in various water samples. The results correlated with those generated using traditional microbial detection methods. (4103)

Notes: 293T cells were seeded in 12-well plates and transfected using 0.4µg of an expression plasmid, 0.6µg of a luciferase reporter plasmid and 60ng of the pRL-SV40 or pRL-TK control vector using FuGENE® 6 transfection reagent. At 24 hours posttransfection, cells were infected with Sendai virus or mouse hepatitis virus, and 8 hours postinfection, luciferase activity was measured using a dual luciferase reporter assay (Promega). (4277)

Notes: The authors investigated the role of the ubiquitin E3 ligase PDLIM2 in the degradation of signal tranducer and activator of transcription 1 (STAT1). They showed that activation of PDLIM2 and subsequent STAT1 ubiquitination require protein kinase C-mediated phoshorylation of PDLIM2 on serine 137. Polyhistidine-tagged PDLIM2 and polyhistidine-tagged mutants PDLIM2-S137A and PDLIM2-S137D were purified using the MagneHis™ Protein Purification System for use in in vitro phosphorylation and ubiquitination assays. One mechanism used to assess levels of STAT1 was a reporter assay using RAW264.7 cells transfected with a pGL3-based construct containing a interferon γ-activated sequence (GAS) upstream of the firefly luciferase reporter gene. Expression of wildtype PDLIM2, but not the mutant forms, resulted in much lower levels of STAT1 protein, and thus lower luciferase activity, when cells were challenged with lipopolysaccharide. The pRL-TK Vector was used to normalize for transfection efficiency. Luciferase assays were performed using the Dual Luciferase^® Reporter Assay System and Reporter Lysis Buffer. (4152)

Notes: The authors investigated the role of pioglitazone on transcriptional regulation of the apoA-I gene and looked at the biological properties of pioglitazone-induced apoA-I-containing high-density lipoprotein particles (HDL). To investigate the biological properties of the HDL particles, the authors treated THP-1 cells with conditioned medium from HepG2 cultures treated or untreated with pioglitazone and looked at adhesion to human aortic endothelial cells (HAEC). During the experiment, HAEC viability and proliferation were monitored using the CellTiter-Glo^® Luminescent Cell Viability Assay. Additionally, to determine whether pioglitazone stimulates apoA-I transcription, a luciferase reporter construct was made containing the apoA-I gene promoter. Transfected cells were treated with pioglitazone, and luciferase expression was monitored using the Dual-Luciferase^® Reporter Assay System. (4173)

Notes: The authors investigated the expression of genes encoding histone-like (H-NS) proteins from the self-transmissible pCAR1 plasmid and Pseudomonas putida KT2440 genome, as well as the interaction of H-NS family members in vitro. Gene expression was quantified using quantitative RT-PCR and RNA templates that were treated with RQ1 RNase-Free DNase to degrade contaminating DNA. Interactions between Pmr and other H-NS proteins were monitored using pull-down assays. His-tagged Pmr was expressed in E. coli, purified and used as bait for FLAG-tagged H-NS family proteins. Protein purification of His-tagged proteins was performed using the MagneHis™ Protein Purification System. (4119)

Notes: The authors investigated the mechanism of microRNA (miRNA)-mediated regulation of both endogenous mRNAs and artificial reporter constructs. To determine whether an m⁷G cap and poly(A) tail are required for repression, the authors created plasmids containing eight synthetic, tandem miR-30 recognition sequences in the 3´ untranslated region (UTR) of a Renilla luciferase gene under the control of a 5´UTR that confers either cap-dependent or cap-independent translation. They used these plasmids as a template for in vitro transcription, then transfected in vitro transcripts with and without an m⁷G cap and poly(A) tail into 293T cells, along with miR-30 miRNA (and miR-21 as a negative control). Similar experiments were performed by transfecting 293T cell with the Renilla luciferase constructs and miR-30 and miR-21 expression vectors. Finally, the authors exchanged the artificial 3´ UTR of the Renilla luciferase construct with the 3´ UTR of BACH1, a transcription factor that is regulated by miR-155, and cotransfected 293T cells with the BACH1 3´ UTR construct and an miR-115 expression vector to examine regulation via an endogenous 3´ UTR. The Promega Primer Extension System was used to compare miR-21, miR-30 and miR-155 RNA levels in transfected and untransfected 293T cells to determine endogenous levels of these miRNAs. Renilla luciferase activity was determined using the Renilla Luciferase Assay System. The authors used Northern blot analysis and quantitative RT-PCR to determine Renilla luciferase RNA levels (and GAPDH levels for normalization purposes). The Plexor® One-Step qRT-PCR System was used for qRT-PCR. (4048)

Notes: The authors expressed the seven subunits comprising the middle module of Mediator, the 1.4MDa coactivator complex required for regulated transcription by RNA polymerase II, in E. coli. To detect and characterize the interaction of these subunits within the module, they coexpressed combinations of these subunits and performed pull-down assays. This enabled them to publish a subunit interaction map. Coexpression and pull-down assays were performed using proteins purified using MagneHis™ Ni-Particles. (4122)

Notes: Listeria monocytogenes uses the internalin A protein (InlA) to cross the intestinal barrier and cause foodborne illness. Mutations in inlA can introduce a premature stop codon, producing a truncated InlA protein that is secreted rather than associated with the bacterial cell wall. Strains with these inlA mutations have reduced virulence. The authors describe an inlA single nucleotide polymorphism (SNP)-based genotyping assay to distinguish isolates with the inlA mutations and use this assay to screen >1,000 L. monocytogenes isolates from ready-to-eat foods and human listeriosis cases. The assay involves amplification of the full-length inlA gene using GoTaq® Colorless Master Mix, purification of amplified products, then single-base-pair extension reactions. (4099)

Notes: The authors studied the ability of JAK1 V658F and A634D mutants to activate the Janus kinase (JAK)/STAT pathway when expressed alone or together with the other components of the interleukin-9 receptor complex. The BOX1 motif of wild-type IL-9Rα, the JAK interacting region, was mutated from PXP to SXS using the GeneEditor™ in vitro Site-Directed Mutagenesis System. To assess STAT transcriptional activity, HEK293 human embryonic kidney, COS-7 monkey kidney, U4C human fibrosarcoma and g2A cells were cotransfected with 250ng of the appropriate constructs, 500ng of firefly luciferase vectors and 50ng of pRL-TK Vector and empty plasmid for a total 1.5µg of DNA. After 24 hours, the cells were lysed in 150µl of Passive Lysis Buffer and reporter activity measured using the Dual-Luciferase^® Reporter Assay System. The ProFection^® Mammalian Transfection System—Calcium Phosphate was used to transfect 10⁶ HEK293 cells in a six-well plate with 3.75µg of plasmid for Western blot analysis and cotransfected 6 × 10⁶ HEK293 cells in a 100mm dish with 14µg plasmid for immunoprecipitation studies. (4025)

Notes: The authors studied the effect of nucleoside modifications in short interfering RNA (siRNA) on 5´ phosphorylation by Clp1 kinase, binding to the Argonaute protein Ago2 and Ago2-mediated cleavage. Mice were injected with 6mg/kg of a siRNA targeting apolipoprotein B (apoB), and total RNA was isolated from the livers after 24 hours. 5´ rapid amplification of cDNA ends (5´ RACE) was used to monitor mRNA cleavage and degradation, as cleaved target RNA yields a 150bp amplification product and uncleaved RNA does not yield an amplification product under the amplification conditions. The amplification steps of the 5´ RACE protocol were performed using GoTaq® Colorless Master Mix and gene-specific primers. (4097)

Notes: To examine post-transcriptional regulation of B cell lymphoma (bcl)-2 mRNA in human cell lines, the bcl-2 open reading frame was cloned into the pCI-neo Mammalian Expression Vector with a FLAG tag. This construct was transfected into U2OS human osteosarcoma cells, lysed, immunoprecipitated on Anti-FLAG-coated beads and analyzed by SDS-PAGE. A 260bp fragment containing the human c-myc 3´UTR adenine-uridine(AU)-rich element (ARE) was cloned into the pGL4.71 [hRlucP] Vector above to produce the pGL4.71PmARE plasmid. For transient expression, SK-N-BE and HEK293 cells in 96-well plates were cotransfected with 200ng of the pGL4.71 constructs (carrying c-myc or bcl-2 ARE) and 200ng of the pGL3-Control Vector. Luciferase expression was assessed using the Dual-Glo^® Luciferase Reporter Assay System. HEK293 cells were also stably cotransfected with the same pGL4.71 constructs and a vector carrying G418 resistance. Renilla luciferase expression was assessed and clones chosen with similar expression levels. (4071)

Notes: The authors studied how the basal rate of protein synthesis in primary cortical neurons was affected by chronic treatment of with a variety of neurotrophic factors and cytokines. Rat eukaryotic elongation factor 2 (eEF2) was cloned by PCR then subcloned into the pCI Mammalian Expression Vector and electroporated into neurons. After 72 hours, the neurons were harvested and used in various translation assays including ribosomal transit time. (4070)

Notes: This study examined the genotype-phenotype correlation of the two major UGT isoforms, UGT1A1 and UGT1A6, involved in resveratrol metabolism. Genomic DNA was isolated from 30mg human liver tissue samples (normal and metastatic) using the Wizard^® SV Genomic DNA Purification System. The purified DNA was eluted with 65°C water and 200–400ng of eluted DNA was used in a PCR-RFLP UGT1A6 genotyping assay. Amplification was carried out using PCR Master Mix in a final volume of 50µl, and the amplimers digested with appropriate restriction enzymes. (4018)

Notes: The authors created a full-length infectious cDNA clone of a genotype 3 hepatitis E virus (strain JE03-1760F) for use in cell culture. The full-length ORF2 sequence of the JE03-1760F genome was amplified and cloned into the pCI Mammalian Expression Vector. The construct was transfected into PLC/PRF/5 cells for 3 days then analyzed by Western blotting. (4029)

Notes: This paper examined the role of the Cdk3/c-Jun signaling in EGF-stimulated cell proliferation and cell transformation. An AP-1 luciferase reporter plasmid construct was contransfected with various expression vector combinations of Cdk3, Cdk2, c-Jun, c-Jun M63/73, Cdk3-DN and cyclin C and the phRL-SV40 Vector as a normalization control. Cells were lysed at room temperature for 30 minutes with gentle shaking and luciferase activity measured using the Dual-Luciferase^® Reporter Assay System. SaoS-2 cells transfected with a mock and Cdk3 RNAi vector were seeded at a density of 4 x 10³ in 96-well plates with 20µl of medium. After 24 hours, 20µl of CellTiter^® 96 AQ_ueous One Solution was dispensed to each well, and the plate incubated for 1 hour at 37°C. The reaction was stopped using 25µl of 10% SDS and absorbance was measured at 492 and 690nm. For the CheckMate™ Mammalian Two-Hybrid System, the plasmid constructs, pACT-c-Jun, pBIND-Cdk3 and pG5luc, were cotransfected into HEK293 cells at no more than 100ng/well in a 48-well plate. After incubation and lysis, 20µl of lysate was dispensed into each well of a 96-well luminescence plate. Firefly and Renilla luciferase activity was detected. (4028)

Notes: The authors tested if CYP1B1 removed cellular oxygenation products that induce oxidative stress and promote the release of antiangiogenic factors. The P450-Glo™ CYP1B1 Assay was used to determine CYP1B1 activity. The presence of glutathione was assessed using either 10⁴ retinal endothelial cells or 50µl of mouse retinal extracts dispensed into each well of a 96-well plate with the GSH-Glo™ Glutathione Assay. (4010)

Notes: Glutathione levels were assessed in human coronary artery endothelial cells (HCAECs) as a measure of oxidative stress. HCAECs were treated with either 100ng/ml eotaxin, a newly discovered chemokine, or pretreated with 2µmol/l MnTBAP for 30 minutes followed by eotaxin treatment for 45 minutes. Positive controls were treatment with 10µg/ml antimycin A and 2ng/ml TNF-α. Cellular glutathione was measured using the GSH-Glo™ Glutathione Assay. (4011)

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)