Notes: This study characterized the strains of hand, foot and mouth disease (HFMD) that circulated in Peninsular Malaysia and Sabah during the last ten years. Viral RNA was extracted from vesicle, throat and rectal swabs, and 5µl of purified RNA was amplified in a 50µl reaction using the AccessQuick™ RT-PCT System with primers for VP4 or VP1. The amplified products were then sequenced and compared to known HFMD sequences. (4341)

Notes: In this paper, FuGENE® HD reagent was used to transiently transfect C2C12 cells using a 4:2 FuGENE® reagent:DNA ratio. (4421)

Notes: These authors evaluated the effect of knockdown of Parp1 on survival of tumor cells after in vivo delivery of anti-Parp siRNA in a lipid-based nanoparticle format. Inhibition of Parp1 expression in mouse tumors was confirmed by qPCR analysis. Total RNA was extracted from the tumors using TRIzol® reagent (Life Technologies) and reverse transcribed using the ImProm-II™ Reverse Transcription System prior to qPCR using SYBR Green PCR Master Mix (Applied Biosystems) . (4222)

Notes: The authors of this study investigated the role of lipid raft microdomains that are present in detergent-resistant membranes (DRMs) during Plasmodium ookinete invasion of A. gambiae midguts. The invasion of the mosquito midgut is a critical step in the Plasmodium life cycle and therefore a promising target of transmission-blocking vaccines. The authors analyzed midgut DRMs by tandem MS and also the glycosylphosphotidyl inositol-anchored subproteome of A. gambiae midgut brush-border microvilli as well. To prepare the GI-anchored proteins for analysis, an in-gel protein digestion protocol was followed using trypsin in the presence of ProteaseMAX™ Surfactant, Trypsin Enhancer. Nearly 97% of the GI-anchored proteins identified were also present in the DRMs, perhaps providing additional targets for transmission-blocking vaccines. (4218)

Notes: To confirm the  Mycoplasma contamination in HIV-1 infected samples detected by high-throughput sequencing, a PCR-based Mycoplasma test was performed. Products were separated on an agarose gel and purified using the Wizard® SV Gel and PCR Clean-Up System before confirmatory sequencing. (4537)

Notes: The authors determined that PERP, a tetraspan membrane protein, is required for enamel formation during tooth development in mice. Using microarray analysis, they then identified genes that were differentially expressed in wildtype and Perp-null mice and might be involved in enamel formation. Differential expression of these genes was confirmed by qPCR using the GoTaq^® qPCR Master Mix. The authors also identified an upstream regulator of Perp, P63, by transfecting cells derived from embryonic mouse teeth with a Perp-luciferase reporter construct that contained a P63 response element or mutated P63 response element. A Renilla luciferase vector was used for normalization of transfection efficiency, and luciferase assays were performed using the Dual-Luciferase^® Reporter Assay System. (4168)

Notes: In this study, HEK293 cells were grown to 50-60% confluence in 100mm dishes and then transfected with plasmid constructs using FuGENE® 6 transfection reagent at a 3:1 FuGENE:DNA ratio. (4363)

Notes: Wizard® SV Gel and PCR Clean-Up System was used to purify products after barcodes were attached by PCR prior to sequencing on an Illumina Genome Analyzer GA2. (4553)

Notes: Pentatricopeptide repeat (PPR) proteins are helical repeat proteins that bind specific RNA segments and protect the adjacent RNA by serving as a barrier to exoribonucleases. This study showed that protection by PPR or PPR-like proteins is the predominant mechanism for defining the positions of processed 5′ and intercistronic mRNA termini in land plant chloroplasts. The authors used RNasin^® Ribonuclease Inhibitor in binding reactions between labeled RNA and PPR proteins prior to Gel mobility shift assays. They also used the pGEM^®-T Vector to clone various 3´ RNA terminal sequences amplified by RT-PCR. (4185)

Notes: The authors used stably transfected HEK cells expressing the human β2 adrenergic receptor, (β2AR) where the C-terminal tail was replaced with the C-terminal tail of the V2 vasopressin receptor (to increase signal-to-noise ratio) followed by a Tobacco Etch Virus (TEV) protease cleavage site and a tetracycline-controlled transcription factor (tTA). A second construct encoding β-arrestin 2 fused to TEV protease was also transfected into the same cell line. To follow the recruitment of β–arrestin upon ligand stimulation of the β2AR, the authors detected the cleavage of tTA and its translocation to the nucleus where it transcribed a stably expressing luciferase reporter gene. The Bright-Glo™ Luciferase Assay Reagent was used to detect luciferase activity and confirm positive recruitment of β-arrestin. The luminescent GloSensor™ cAMP Assay was used to assess ligand stimulation of the β2AR signaling through G proteins, resulting in an increase in cAMP. The authors used both of these assays to quantify ligand bias and identify weakly biased compounds of the β2AR and angiotensin II type 1A receptors. A number of known compounds were assessed to show the value of this strategy in helping to decipher complex signaling pathways. This approach may be useful in the development of novel biased ligands for therapeutic use. (4146)

Notes: The full-length 3´ UTR of mouse RNA-binding protein HuR was amplified and cloned into psiCHECK™-1 Vector to create pEM429 plasmid. To generate radiolabeled RNA substrates for use in cleavage assays, RNA was synthesized from 1µg of linearized plasmid using 50µCi of [α-³²P]UTP, 0.8mM Ribo m⁷G Cap Analog and T7 RNA Polymerase by incubating for 1.5 hours at 37°C. The RNAs were then treated with 1 unit of RQ1 RNase-Free DNase per 1µg of template DNA for 15 minutes at 37°C before extracting with phenol:chloroform, precipitating with ethanol and resuspending in DEPC-treated water. The cleavage assay used 60fmol of ³²P-labeled substrate RNA with 10U Recombinant RNAsin Ribonuclease Inhibitor in a reaction incubated for 2.5 hours at 30°C. RNA probes were labeled with biotin using T7 or T3 RNA Polymerases with a biotin-UTP labeling NTP mixture and incubated for 2 hours at 37°C. The biotinylation reaction was then treated with RQ1 RNase-Free DNase following the same protocol used for radiolabeled RNA. To form HuR/RNA complexes, 2µg of biotinylated RNA was mixed with 100µg nuclear extract and 40 units Recombinant RNAsin^® Ribonuclease Inhibitor in a total volume of 20µl, and incubated for 30 minutes at room temperature. For CstF-64/RNA complexes, 1µg of biotinylated RNA was used and the complexes were stabilized by UV crosslinking using 10U Recombinant RNAsin Ribonuclease Inhibitor during the UV treatment. NIH 3T3 cells were UV crosslinked and either total or nuclear RNA used for immunoprecipitation. After extraction the RNAs were treated with RQ1 RNase-Free DNase for 15 minutes at 37°C before RT-PCR using HuR-specific primers. Total RNA purified from cultured cells were incubated with 50U/ml RQ1 RNase-Free DNase at 37°C for 30 minutes before use in RT-qPCR. (4187)

Notes: In this study, FuGENE® 6 Reagent was used to transiently transfect the S2R+ drosophila Schneider cell line with 0.6µg DNA at a 8.3:1 FuGENE:DNA ratio. 0.6 x 10e6 cells were plated 1 day prior to transfection in 12-well plates. (4369)

Notes: These authors investigated whether HaloTag® technology could be used to specifically target and degrade intracellular proteins. They developed a method to degrade specific proteins of interest using a small molecule by tagging the HaloTag® protein with an adamantyl moiety. The hypothesis was that appending a hydrophobic residue to a protein surface domain would mimic partial denaturation and induce proteasomal degradation. They first used a HaloTag®-luciferase fusion protein to determine the biological activity of various small molecules that enhanced hydrophobicity. After selecting the small molecule that reduced luciferase activity in HEK293 cells, they went on to test their hypothesis in various in vitro and in vivo models. They were able to demonstrate degradation of various HaloTag®-linked transmembrane proteins expressed in HEK293 cells, to show degradations of target proteins in Zebrafish, and to show degradation of the HRAS oncogene product both in NIH3T3 cells and in a mouse tumor model. (4125)

Notes: These authors compared the performance of automated DNA purification using the Maxwell® 16 Instrument with manual methods. DNA yield and quality from milk, blood (buffy coat), and semen samples from cattle were evaluated. The Maxwell® 16 System performed best, consistently generating high quantity and quality DNA across the sample range tested. (4213)

Notes: This study explored whether the Agr-like quorum-sensing system regulates sporulation and production of the Clostridium perfringens toxins CPE and beta2 toxin. An agrB null mutant was inhibited for production of beta2 toxin during vegetative growth and in sporulating culture, had reduced production of alpha-toxin and perfringolysin O during vegetative growth, and did not form spores efficiently. The AccessQuick™ System was used in RT-PCR analysis to confirm the presence or absence of agrB transcripts in the wild type, mutant, and complemented strains used in the study. (4546)

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)