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 vasculoproliferative disorder peliosis hepatis has been linked to Bartonella henselae infection in humans and dogs. The authors sought to determine if this is true in cats, the natural reservoir for B. henselae, using immunohistochemistry (IHC) and PCR. Tissue sections from 26 cats with peliosis hepatis were formalin-fixed and paraffin-embedded and subjected to IHC using a B. henselae-specific antibody. To detect B. henselae DNA, PCR was performed using GoTaq® Flexi DNA Polymerase, genus-specific primers for the pap31 gene of Bartonella, 1mM MgCl₂ and total DNA isolated from 8µm paraffin-embedded tissue sections. The presence of Bartonella DNA was confirmed by PCR using species-specific primers that target the B. henselae heat-shock protein (htrA). These studies found no link between B. henselae infection and peliosis heptis in cats. (4094)

Notes: The authors compared seedling growth rates, photosynthesis rates and expression levels of heat-responsive genes in the heat-resistant wild rice Oryza meridionalis and the domesticated rice O. sativa when grown at optimal and elevated temperatures. Proteins that were up- or downregulated in response to heat were identified by two-dimensional gel electrophoresis coupled with nano liquid chromatography on line with tandem mass spectrometry (nanoLC-MS/MS). Trypsin was used to cleave proteins prior to nanoLC-MS/MS. Semi-quantitative RT-PCR was performed using the GoTaq® Green Master Mix to determine if the heat-responsive proteins were transcriptionally regulated. (4092)

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 microtubule-based cytoskeleton of Toxoplasma gondii is important for cellular invasion. The authors of this paper investigated the tubulin composition and structure
and showed that T. gondii tubulin is extensively altered by post-translational modification. These modifications were analyzed by mass spectrometry. Trypsin digestion of dried gel pieces containing tubulins was performed overnight at 37 °C with the ProteaseMAX Surfactant trypsin enhancer. (4082)

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: As part of this study, the authors performed in-gel digestion of expressed protein fragments using trypsin and ProteaseMax Surfactant. The protein of interest was first run on an SDS-PAGE gel and the band excised, cut into 1 x 1mm pieces, reduced with 50 mM DTT and alkylated with 100 mM acrylamide. After destaining with 50 mM 1:1 acetonitrile:ammonium bicarbonate solution, the samples were dried and reconstituted in 12 ng/μL trypsin in the presence of ProteaseMax. The mixture was incubated at 50°C for 1 h before being spun down at 12g for 30 s. The isolated peptides were dried, then reconstituted in 10 μL of 2% acetonitrile/water with 0.1% formic acid before LC-MS/MS analysis. (4079)

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 characterized mouse neocortical interneurons that express 5-HT_3A, a ligand-gated cation channel activated by 5-hydroxytryptamine (serotonin), during embryonic development. Transgenic mice that expressed green fluorescent protein (GFP) under the control of the 5-HT_3A promoter were created. Single 5-HT_3A-expressing neurons within 300µm brain sections of transgenic mice at various stages of embryonic development were subjected to whole-cell path-clamp recordings to examine their electrophysiological properties. To confirm activation of the 5-HT_3A promoter in these cells, GFP expression was visualized by fluorescence microscopy without breaking the patch clamp seal. The contents of these single neurons then were aspirated and expelled into a 10µl reverse transcription reaction. After the reverse transcription, PCR was performed to simultaneously detect mRNAs encoding two isoforms of glutamic acid decarboxylase, three calcium-binding proteins, three neuropeptides, two transcription factors and reelin, a protein thought to be involved in neuronal migration and morphology. Two rounds of PCR using nested primers were required to detect these mRNAs. PCRs were performed using GoTaq® DNA Polymerase. Amplified products were visualized by agarose gel electrophoresis, using the 100bp DNA Ladder as a size standard. (4096)

Notes: In this paper, FuGENE® 6 or HD reagent was used to transiently transfectCHO-K1 cells. Transfection conditions were as follows: Cells were plated in 60mm dishes and transfected with FuGENE® 6 using 1.03µg of DNA. Cells were plated in 100mm dishes and transfected with FuGENE® 6 or FuGENE® HD using 6µg of DNA. 

Notes: In this paper, the authors were interested in the proteomic analysis of subcellular compartments to see if there were any protein markers for colorectal cancer (CRC) when analyzing early stage tumors and colorectal adenoma and carcinoma tissues. CRC tissue was collected and assessed for microsatellite instability and chromosomal copy number changes using the MSI Analysis System, Version 1.1, and MLPA respectively. The tumor proteins then were analyzed using MALDI-TOF/TOF MS. (4116)

Notes: Human Genomic DNA:Male was used as a negative control in standard PCR and Sanger Sequencing to confirm fusion genomic breakpoints identified by NGS experiments. (4534)

Notes: The authors identified proteins that bind to small ubiquitin-like modification (SUMO) proteins using a yeast two-hybrid screen. The proteins ability to bind SUMO was confirmed using protein:protein interaction studies. In these studies, recombinant SUMO-1 was expressed with a His₆ tag and immobilized using MagneHis™ Ni-Particles. Putative SUMO-binding proteins were expressed with a GST tag and the proteins were co-incubated to allow them to interact. Proteins bound to immobilized SUMO-1 were eluted with Laemmli sample buffer and detected by Western blot. (4121)

Notes: The Wizard® SV Gel and PCR Clean-Up System was used to purify PCR products prior to pyrosequencing. (4546)

Notes: The authors used the PowerPlex® Y System to amplify Y-STRs from 154 sexual assault cases in the Philippines. Duplicate amplifications were assembled as recommended by the manufacturer, and amplified products were detected using an ABI PRISM® 310 Genetic Analyzer. Microscopic analysis revealed sperm in 15% of the cases, and Y-STR analysis revealed male DNA in 41% of the cases. (4088)

Notes: FuGENE HD reagent was used to transiently transfect U87MG cells using a 5:2 ratio of reagent to DNA (5µl reagent, 2µg DNA). Following transfection, cells were incubated for 48 hours before use. (4412)

Notes: These authors studied the formation of centrioles in Drosophila spermatids. Genomic DNA was extracted from whole flies using the Wizard^® SV Genomic DNA Purification System. The DNA was then subjected to PCR, purified and sequenced. RNA was purified from whole flies using the SV Total RNA Isolation System. After RNA extraction, the samples were used in RT-PCR. (4064)

Notes: These authors describe a method for comprehensive expression profiling of tissue mitochondria, using swine heart as an example. After protein extraction, a 2-step trypsin digestion method was used to obtain peptides. The authors evaluated use of ProteaseMax as an alternative method to enhance digestion during the first digestion step of the process. The advantages and disadvantages of the method are discussed. (4085)

Notes: Mineral accumulation was studied in Arabidopsis thaliana comparing loci involved with growing in soil versus hydroponics. An F2 population derived from a cross between Landsberg erecta (Ler; maternal parent) and Eringsboda-1 (Eri-1; paternal parent) was propagated by single seed descent for nine successive generations in soil.
The flower buds of three plants per line were collected, and the DNA extracted using the Wizard® Magnetic 96 DNA Plant System and used for genotyping with 90 amplified fragment length polymorphism PCR (AFLP) and 39 single sequence length polymorphisms (SSLP) markers to build a genetic map of quantitative trait loci (QTL). (4136)

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)