Notes: The authors investigated starch synthesis in barley (Hordeum vulgare) by examining mutations in class I, class II and class III starch synthases (ssI, ssII and ssIII, respectively). Mutations of ssIIa and ssIIIa were detected by PCR using the GoTaq^® Hot Start Polymerase. (4162)

Notes: Wizard® SV Gel and PCR Clean-Up System was used to extract and purify DNA fragments that had been amplified by long-range PCR and separated on an agarose gel prior to next-generation sequencing on an Illumina platform. (4535)

Notes: The authors used the Dual-Luciferase® Reporter Assay System to measure NF-κB firefly luciferase activity normalized to Renilla luciferase (pRL-TK) in transfected HEK293T and EL4 cells. Coumermycin-treated HEK293 and EL4 cells transiently transfected with B-cell CLL/lymphoma 10 (BCL10) led to transcriptional NF-κB activation in a dose dependent manner. Dimerization of BCL10 by coumermycin was used to mimic physiological stimulation through T cell receptor cross-linking, which initiates BCL10-mediated activation of the NF-κB signaling pathway. Overexpression experiments showed that E3 Ubiquitin Ligase Mind Bomb-2 (MIB2) controlled BCL10-mediated activation of NF-κB by promoting autoubiquitination and ubiquitination of IKKγ, a kinase responsible for phosphorylating IκBα protein. The authors identified that the C-terminal RING finger domain of MIB2 was critical for protein ubiquitination in NF-κB activation, which was confirmed by the NF-κB luciferase reporter response to various MIB2 mutants. (4180)

Notes: The authors looked at changes in the rice leaf proteome to understand the molecular mechanisms involved in silicon-induced cadmium tolerance. Total protein was extracted from leaves of plants grown in the presence of sodium silicate or cadmium sulfate. Proteins were separated on 2-D gels, and protein spots were manually excised, reduced and alkylated before digestion with trypsin in the presence of ProteaseMAX™ Surfactant, Trypsin Enhancer. Digests were analyzed via MALDI-TOF MS analysis. The researchers identified 60 proteins, 50 of which were regulated by silicon. (4217)

Notes: These authors first used a FLAG-tagged protein (nfr2) with a HeLa Nuclear extract and captured interacting proteins via SDS-PAGE and in-gel digests of bands to identify (Krüppel-associated box)-associated protein 1 (KAP1) as a potential interacting partner. Human KAP1 was purchased as a HaloTag^® CMV Flexi^® Vector from Kazusa and used in a Mammalian PullDown scenario (with HaloLink™ Resin) to demonstrate interaction between the two proteins. A reporter assay was used to show that KAP1 facilitates Nrf2 transactivation in a dose-dependent manner. The authors defined the interaction sites using GST-tagged nrf2 and various forms of KAP1-HaloTag® Fusions expressed in TNT® SP6 High-Yield Wheat Germ Extract. GST-tagged proteins were expressed in E. coli and bound to glutathione-Sepharose beads. These bound proteins were mixed with the KAP1 from the cell-free expression system, incubated for 4 hours at 4°C, washed and stained with the HaloTag^® TMR Ligand for 30 minutes. The proteins from the pull-down assay were subjected to SDS-PAGE and the HaloTag^® proteins detected by phosphorimaging and the GST proteins by Coomassie Brilliant Blue Staining. A two-hybrid system consisting of the pRL-TK Vector with a firefly luciferase reporter with Gal4 UAS, mouse Nrf-2 N-terminal domain and KAP1 was also used. The vectors were transfected into Nrf2 knockout MEFs for 4 hours then incubated for 36 hours before luciferase expression was determined using the Dual-Luciferase^® Reporter Assay System. (4123)

Notes: Proteasome activity was determined in cells expressing the β2 proteasome subunit using the Proteasome-Glo™ Trypsin-Like Cell-Based Assay. The authors determined the proteasome activity in the cells after exposure to the proteasome inhibitor MG132. Luminescence indicative of proteasome activity was measured using a GloMax^®-Multi Detection System Luminometer. (4195)

Notes: To investigate if house flies could act as a vector for avian influenza virus H1N5, flies exposed to the virus were homogenated and the homogenate used to inoculate 10-day-old embryonated chicken eggs (ECEs). Allantoic fluids were collected from the eggs, RNA purified from the samples and the presence of H1N5 assessed using M-specific primers and the AccessQuick™ RT-PCR System and looking for the 276bp amplimer. (4339)

Notes: HEK293T adenovirus-transformed human kidney cells were plated in 10cm plates with 5 × 10⁶ cells or in 12-well plates with 3 × 10⁵ cells/well before transfection using with 5µg or 0.2µg of viral reporter plasmids, respectively. To transfect the cells, FuGENE® 6 Transfection Reagent was used at a ratio of 2.5µl:1µg of reagent:DNA. (4401)

Notes: Thrombomodulin (TM) expression was examined by isolating genomic DNA from biopsies of human malignant mesothelioma and normal mesothelial tissue, and cultured cell lines with or without PARP1 silencing treated with 5-aza-2´-deoxycytidine and trichostatin alone or in combination and then subjected to biosulfide modification. To analyze methylation of TM, a CpG island in the promoter, 5´ UTR and an exon region containing 44 CpG dinucleotides were PCR amplified, cloned into the pGEM^®-T Easy Vector, transformed and positive clones selected using IPTG/X-Gal and analyzed by PCR. Colonies were cultured, the plasmids isolated using the Wizard^® Plus SV Minipreps DNA Purification System then 10 clones
from each sample type were sequenced. (4132)

Notes: Fugene HD was used in a transient transfection of HeLa cells with 0.5µg of DNA in a ratio of 3.5:1 transfection reagent to nucleic acid. Cells were plated 16 hours before transfection at a density of 0.9 × 10⁵ cells/well in a 24-well plate. (4409)

Notes: The authors of this study set out to describe the mechanism of cell death through which TNF-like weak inducer of apoptosis (TWEAK) exerts its apoptotic effect on certain cancer cells. The used the CellTiter-Glo® Cell Viability Assay and the Caspase-Glo® 3/7 Assay to investigate cell viability and mechanism of cell death in HSC3 cells treated with TWEAK. They looked at caspase-8 activity in cells treated with TWEAK in the presence or absence of a caspase-8 inhibitor using the Caspase-Glo® 8 Assay. They showed that TWEAK induces caspase-dependent apoptosis in these cells. (4170)

Notes: pGL4.35  and pGL4.36 reporter vectors were used. (4264)

Notes: The authors used Dual-Glo^® Luciferase Assay to measure a pISG54 promoter-driven firefly luciferase gene (0.5µg) and pRL-TK plasmid constitutively expressing Renilla luciferase (0.1µg), and either a plasmid (0.5µg) expressing the corresponding variants of Ebola virus (EBOV) structural protein VP24 construct (phCMV-EBOV-VP24) or empty phCMV in HEK 293T and GPC-16 transfected cells. A total of 1.1µg of DNA was transfected. Cells were stimulated with interferon (IFN) 24 hours post-transfection, harvested 16 hours later, and assayed for dual-luciferase activity. Data indicated that mutations in the V24 protein were associated with EBOV virulence but that this virulence was not linked to the IFN-antagonist function of V24 protein. (4178)

Notes: RNasin was used in the small RNA library preparation step before sequencing on an Illumina platform.

Notes: The authors of this paper investigated the mechanism of interaction of HIV-1 Integrase with host cell DNA. To understand the role of the integrase Zn-binding domain, they studied the effect of the Zn ejector 2,2'-dithiobisbenzamide (DIBA) on DNA binding. The covalent modification of integrase by DIBA was evaluated using in-gel tryptic digestion and SELDI mass spectrometry analysis. ProteaseMAX Surfactant was used to enhance protein digestion. (4080)

Notes: The authors transiently transfected 10⁵ HMC-1 cells per well of a 24-well plate using the FuGene® HD Transfection Reagent, 0.5µg of DNA and a reagent:DNA ratio of 4:1. (4429)

Notes: The authors determined that a recombinant catalytic domain of rat matriptase (His₆t-S-CD) caused detachment of small-intestinal epithelial cells (IEC-6 cells) from laminin-coated plates. His₆t-S-CD was expressed in the yeast P. pastoris and purified by ammonium sulfate precipitation, gel filtration through a PD-10 column, then Ni²⁺-chelating chromatography using HisLink™ Resin. The authors also treated IEC-6 cells with purified His₆t-S-CD to determine if this domain induced apoptosis by monitoring annexin-V staining, DNA fragmentation and caspase-3 activity. For the DNA fragmentation analysis, IEC-6 cells were treated with His₆t-S-CD, then harvested, and genomic DNA was purified using the Wizard® SV Gel and PCR Clean-Up System. DNA fragmentation was assessed by agarose gel electrophoresis and ethidium bromide staining. (4100)

Notes: The authors created two new osteosarcoma models expressing the firefly luciferase enzyme, using a modified pGL3 plasmid to insert luciferase into lentiviral particles for transfection. Luciferase activity was detected using Steady-Glo® Luciferase Assay System with varying numbers of osteosarcoma cells in 96-well microplates. The luciferase-expressing osteosarcomas showed conserved osteolytic and osteogenic activities in mice, and could be imaged in vivo. The authors developed protocols to administer small interfering RNA to its osteosarcoma target (luciferase), and demonstrated its efficiency in vivo to suppress luciferase expression as a model for siRNA treatment of cancer. (4127)

Notes: The authors identified a single nucleotide polymorphism (SNP) in the 3´ untranslated region (3´ UTR) of MDM4, which promotes tumorigenesis by decreasing p53 tumor suppressor function, in ovarian cancer cells. This A to C transversion creates a putative target site for the hsa-miR-191 microRNA in the MDM4-C allele, but not the wildtype MDM4-A allele. To determine if this SNP affects MDM4 translation efficiency or mRNA stability, the authors cloned a 224-bp fragment of the MDM4 3´UTRs into the psiCHECK™-2 Vector and transfected the ovarian cancer cell line A2780 with the 224A or 224C variants of the MDM4 3´ UTR. The authors used the luciferase-based psiCHECK™-2 Vector and Dual-Glo^® Luciferase Assay System to show that the C variant dramatically reduces translation efficiency and/or mRNA stability. They also assessed MDM4 expression levels in A/A, A/C and C/C ovarian cancer cells and tissues using RT-qPCR. qPCR primers for MDM4 were designed using the Plexor^® Primer Design Software, and assays were performed using the Plexor^® qPCR System. (4157)

Notes: Wizard® SV Gel and PCR Clean-Up System was used to clean up genomic DNA isolated from parasitic nematodes isolated from a variety of animals. Species identification of each nematode specimen was determined via PCR amplification of specific nuclear DNA followed by purification of the amplified product using Wizard® PCR Preps DNA Purification System before sequencing using BigDye chemistry. Wizard® SV Gel and PCR Clean-Up System was also used to prepare amplicons generated by long-PCR of mt genomes from the nematodes before NGS sequencing. Results from NGS were confirmed using PCR-based sequencing of short mt DNA tracts. Short mtDNA regions were amplified by conventional PCR. Amplicons were purified using Wizard® PCR Preps DNA Purification System before sequencing using BigDye chemistry. (4533)

Notes: The authors explore barley (Hordeum vulgare) as a means of expressing recombinant human Flt3 ligand, which is a growth factor involved in proliferation and differentiation of stem cells and development of various immune cells. As part of their quality control, they performed in-gel proteolytic digestion and mass spectrometry. The recombinant Flt3 ligand was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Coomassie® blue staining, excision of the protein band, destaining, drying of the gel slice and digestion with Sequencing Grade Modified Trypsin at 30°C overnight prior to mass spectrometry. To test biological activity, the authors treated human acute myeloid leukemia cells with Flt3 expressed in barley or a commercial source of Flt3 then measured cell proliferation using the CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay. (4352)

Notes: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) tends to cause apoptosis in tumor cells over normal cells, and so the TRAIL ligand and signaling pathway is an attractive pathway for developing cancer therapeutics. Bortezomib is a proteasome inhibitor that is used for therapy in many cancers. Studies indicate that it can sensitize tumor cells to the apoptotic effects of TRAIL. In this study, the authors investigated the ability of bortezomib on renal cell carcinoma (RCC). Growth inhibition of RCC in response to treatment with bortezomib followed by TRAIL treatment in the presence or absence of caspase inhibitors was assessed using the CellTiter® 96 AQ_ueous Non-Radioactive Cell Proliferation Assay. They assessed caspase activity in bortezomib/TRAI- treated RCC using the Caspase-Glo® 8 Assay. The effect of bortezomib on the proteasome of the RCCs was investigated using the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay. Their studies suggest that bortezomib can sensitize some RCC to TRAIL signaling. (4169)

Notes: The authors used microarray analysis to identify genes that are differentially expressed in nectaries of Arabidopsis and may be involved in nectar synthesis and secretion. One of these genes was cell wall invertase 4 (cwinv4). The authors generated two Arabidopsis cwinv4 mutant lines to study gene function and used GoTaq® Green Master Mix to confirm the mutant genotype. Expression patterns of cwinv4 in wildtype Arabidopsis and an ortholog from Brassica rapa were examined in various tissues by RT-PCR. The reverse transcription step was performed using the Reverse Transcription System and 0.1µg of RNA. (4093)

Notes: The authors investigated genetic diversity in a Florida population of Illicium parviflorum, an endangered evergreen shrub, by amplifying intersimple sequence repeats (ISSRs). Amplifications were performed using GoTaq^® Hot Start Polymerase. (4161)

Notes: These authors investigated the role of the cardiac lineage protein 1 (CLP1/HEXIM1) in skeletal myogenesis. They showed that CLP1 knockout C2C12 cells were unable to differentiate, and then investigated the hypothesis that CLP1 associates with MyoD and HDAC proteins to downregulate cell cycle genes, such as cyclin D1, and allow expression of differentiation-specific genes. RNasin® Ribonuclease inhibitor was used in coimmunoprecipitation assays investigating the interaction between CLP1 and HDAC in C2C12 cells under differentiation and non-differentiation culture conditions. RNasin® was included during cell lysate preparation prior to coimmunoprecipitation assays with antibodies directed against various HDAC proteins. The authors also performed  a luciferase reporter assay using the Dual-Luciferase® Assay System to investigate the regulation of cyclin D1 expression by CLP1, MyoD and HDAC. A luciferase reporter-cyclin D1 construct was co-transfected with MyoD, HDAC and CLP1 constructs and the effect on luciferase expression examined under differentiation and non-differentiation conditions. In differentiation medium MyoD, CLP1 and HDAC5 acted synergistically to reduce cyclin D1 expression. (4225)