Notes: Researchers were interested in determining if knockdown of cellular FLICE inhibitory protein (c-FLIP) would affect caspase-8 activation. Instead of assaying cultured cells for the Caspase-Glo^® 8 Assay, the authors used siRNAs targeting c-FLIP_L, c-FLIP_S or nontarget control to transfect A549 cells. Equal number of cells were harvested and treated with FLAG-tagged Apo2L/TRAIL + anti-FLAG M2 antibody at 37°C for various times prior to lysis. The lysate was immunoprecipitated overnight using protein A/G beads. After washing with lysis buffer, the beads were resuspended in phosphate buffer and the immobilized death-inducing signaling complex (DISC) was assayed for caspase-8 activity in 96-well plates using an equal volume of Caspase-Glo^® 8 Reagent.
(3277)

Notes: Researchers compared MagneHis™ Ni-Particles to other vendors’ his tag protein purification systems in a model co-precipitation system with various bait proteins and a Shewanella oneidensis degradosome. Peptides from the S. oneidensis degradosome that co-purified with the bait proteins were analyzed by SEQUEST analysis. Bait proteins included E. coli polynucleotide phosphorylase, (PNP), RNase E and the RNA helicase. The authors describe the use of the MagneHis™ Ni-Particles in a simple and efficient system that can be automated for screening purposes. The authors also discussed optimizing the elution conditions, amount of bait protein and wash steps. (3280)

Notes: To identify novel regulators of the HIF-1 pathway, the authors performed a knockdown experiment using a synthetic siRNA library against a number of kinases. Two of their ‘top hits’ directly down-regulated the hif-1a mRNA through a 7nt complementation. They built an siRNA library using an HIF-1 reporter assay. For siRNA library screening, the HIF-1 reporter and a control reporter (pRL-TK Vector, Promega) were transfected into H1299 cells. The Dual-Glo™ Luciferase Assay System was used to analyze cells transfected with an siRNA against HIF-1a as a positive control and an siRNA irrelevant to the HIF-1 pathway as a negative control. (3538)

Notes: The human lung cell line A549 and the human liver cell line were transiently transfected with 0.1µg of CYP2F1 reporter constructs and 0.001µg of pRL-TK Vector using FuGENE® 6 Reagent in 96-well plates. For cotransfection studies, cells were transfected with 0.1µg of the reporter construct, 0.002µg of pRL-TK plasmid, and 0.5 or 0.2µg of Sp1, Sp3 or empty expression vectors, with the total transfected DNA remaining at 0.2µg. Reporter activity was assessed using the Dual-Luciferase® Reporter Assay System.

SL-2 cells were seeded in 24-well plates and cotransfected with 0.3µg of CYP2F1 reporter plasmid and 0.3µg of pPac/Sp1, pPac/Sp2 or empty expression vector. The total amount of plasmid DNA used for each transfection was 0.9µg. The DNA and FuGENE® 6 were added at a 3:1 ratio. Activities were assessed using the Dual- Luciferase® Reporter Assay System.

The Gel Shift Assay System was used to identify Sp1-like sites in the promoter of the human CYP2F1 using EMSA (electromobility shift analysis). (4269)

Notes: The authors of this study investigated the role of Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1) in Toll-like receptor 4 (TLR4)-mediated lipopolysaccharide (LPS) response. RAW264.7 macrophages were transfected with wildtype or mutant SHIP1 or control plasmid. Anti-ACTIVE^® JNK and p38 antibodies were used in Western analyses to determine phosphorylation of these kinases in response to overexpression of SHIP1 or mutant SHIP1. To investigate any effects on IκB-alpha and NF-κB expression, RAW264.7 macrophages were cotransfected with pGL3-XκB-luciferase reporter plasmid and pRL-TK Renilla luciferase control plasmid. Transfected cells were treated with LPS for 6 hours or left untreated (control). The Dual-Luciferase^® Reporter Assay System was used to monitor reporter gene expression. (3524)

Notes: To test the activity of torI as a prophage excisionase, the chloramphenical aceytltransferase gene was introduced into a non-coding region of the prophage KplE1 in a strain of E. coli containing a torI-encoding plasmid. Expression of torI was induced by IPTG and the cells plated on either chloramphenical or ampicillin. GoTaq^® DNA polymerase was used in a PCR test to confirm prophage DNA excision from randomly chosen ampicillin-resistant colonies. (3329)

Notes: GoTaq^® Polymerase was used in this study investigating the role of TorI (Tor inhibition protein) in prophage DNA excision. PCR products were amplified from E. coli strains carrying a plasmid-encoded torI gene, and were analyzed by gel electrophoresis. (3350)

Notes: To identify proteins expressed on the cell surface of various strains of Leptospira, the authors labeled intact Leptospira cells with biotin, solubilized the cells with Triton^® X-100, then captured the biotinylated proteins using the SoftLink™ Soft Release Avidin Resin. The captured products were analyzed by one- and two-dimensional gel electrophoresis. The spots were excised and washed with 50mM ammonium bicarbonate /100% acetonitrile. The gel pieces were then dried, rehydrated in a solution containing 12ng of Sequencing Grade Modified Trypsin, and incubated in 50mM ammonium bicarbonate overnight at 37°C. The proteins were concentrated, desalted and subjected to MALDI-TOF mass spectrometry to identify the individual proteins of the Leptospira surfaceome. (3661)

Notes: These authors characterized metabolic pathways in Borrelia burgdorferi, the causative agent of Lyme disease, focusing on the 5´-methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs) and the autoinducer-2 production protein LuxS. Recombinant LuxS and Pfs proteins from both B. burgdorferi and E. coli were expressed as polyhistidine-tagged proteins in BL(21)DE3pLysE and purified using the MagneHis™ Protein Purification System. The purified proteins were then used in enzyme activity assays. The E. coli LuxS and Pfs proteins were used as positive controls for enzyme activity. (3566)

Notes: The Wizard^® SV Genomic DNA Purification System was used to purify total DNA from plasmid-electroporated EBV-positive D98-HR1 cells. The isolated DNA was digested by two different restriction enzyme combinations and used in Southern blots to identify electroporated plasmid levels. (3231)

Notes: In this study, RAW 264.7 mouse macrophage, Abelson murine leukemia virus-induced tumor cells and HepG2 human liver hepatocellular carcinoma cells were transfected using FuGENE® 6 Transfection Reagent. Cells (3–5 × 10(5) RAW 264.7 cells and 2 × 10(5) HepG2 cells plated/well of a 12-well plate) were transfected with DNA at a 3:1 FuGENE:DNA ratio; the transfection mix included 0.05µg of control vector. (4365)

Notes: In this study, the binding of the response regulator RR06 to the promoter region of cbpA was investigated. DNA encoding the rr06 gene was cloned into the pGEM^®-T Easy vector and transformed into E. coli strain DH5α. Cell lysates were prepared and incubated with a labeled cbpA promoter fragment, and binding was evaluated in an electrophoretic mobility shift assay (EMSA). To further investigate the RR06/cbpA interaction, the cbpA promoter region, or a control rRNA sequence, was biotin labeled and attached to Streptavidin MagneSphere^® Paramagnetic Particles. The beads were incubated with E. coli lysates expressing RR06, or with control lysates. After washing, bound proteins were eluted by boiling. Eluted samples were then probed with specific anti-RR06 antisera. (3397)

Notes: A fragment containing HIV-1 LAI 5' LTR was cloned into the unique EcoICRI-XhoI site of the pGL3-Basic Reporter Vector. The Luciferase Reporter Assay was used to analyze DNA transfected cells for luciferase activity. The pRL-TK Vector was used as a transfection efficiency internal control with a Renilla cDNA under control of HSV-TK. Firefly luciferase activity derived from the HIV-1 LTR was normalized to the Renilla luciferase activities using the Dual-Luciferase® Reporter Assay. (3703)

Notes: To determine the potential role that T-bet, a T-box transcription factor that specifies Th1 lineage commitment, and Hlx, a homeobox gene, may have in helper T cell differentiation, the physical interaction between the two proteins was tested using the CheckMate™ Mammalian Two-Hybrid System. The pACT-T-bet and pBIND-Hlx fusion vectors were co-transfected with pG5luc into 293T cells. After 48 hours, the firefly and Renilla luciferase activities were measured using the Dual-Glo^® Luciferase Assay System. (3493)

Notes: To analyze whether CD8⁺ T cell responses can be generated from gene products translated from another reading frame from the primary ORF encoded by DNA vaccines, various constructs were made using the pCI Mammalian Expression Vector. These inserts included the small hepatitis B surface antigen (HBsAg) and its 3’ untranslated sequence up to the hepatitis B virus (HBV) poly(A) sequence, which contains the sequence encoding the C-terminal Pol_344–832 fragment; the 832 residue Pol protein, and the large HBsAg in an alternative reading frame and an unrelated in frame and out of frame OVA OVA_18–385 fragment. In the case of the OVA fragment, the CMV promoter was removed and substituted with the SV40 promoter from pSI Mammalian Expression Vector. The immune response to both the primary and secondary gene products was determined using transient transfection of LMH cells followed by Western blotting or immunoprecipitation or counting CD8⁺ T cells from spleen cells. (3553)

Notes: The Kinase-Glo^® assay was used to assess Cdk4 kinase activity in HeLa cell lysates. In these experiments, 100μg of lysate (by protein determination) were immunoprecipitated using 1μg of a mouse monoclonal anti-Cdk4 antibody and 20μl protein G sepharose beads. The immobilized Cdk4 kinase immunoprecipitates were then washed three times in lysis buffer and twice in kinase buffer (50mM, pH 7.5, 10mM MgCl₂, 250μM EGTA, 10mM β-glycerophosphate, 100μM Na₃VO₄, 1mM DTT), and resuspended in 30μl of kinase buffer. Cdk4 Kinase activity was then assessed by incubating the immunoprecipitates with 20μl of 0.1μM ATP and 2μg Rb peptide for 30 minutes at 30°C. After this incubation the Kinase-Glo^® Detection Reagent was added and the resulting luminescence recorded. Data are presented as relative luminescence from various samples. (3304)

Notes: These authors investigated the mechanisms by which anti-carcinogenic compounds derived from Japanese and subtropical vegetables and fruits attenuate LPS-induced COX-2 mRNA expression in RAW264.7 mouse macrophages. Using Western blot analysis, 10µg nuclear or 20µg cytosolic protein fractions isolated from LPS-treated macrophages in the absence or presence of various phytochemicals were stained with several antibodies for signal pathway proteins, including the Anti-ERK1/2 pAb. Further analysis of the MAPK and NF-κB systems was performed using firefly luciferase constructs co-transfected with the control pRL-TK Vector at a 1:1 ratio. Transfected cells were exposed to the plant compound for 12 hours and then to LPS for a further 12 hours. Reporter activity was measured using the Dual-Luciferase^® Reporter Assay System. To determine the effect of the phytochemical zerumbone on the kinase reaction, a cell-free kinase assay was performed using Kinase-Glo^® Assay System. In this assay, 1.5µl recombinant MAPKAPK-2, 0.2µl recombinant active p38α, 1µl zerumbone and 4µmol/l ATP were incubated for 3 hours at 28°C before addition of an equal volume of Kinase-Glo^® Reagent. (3333)

Notes: The authors performed a yeast two-hybrid screen using big mitogen-activated kinase 1 (BMK1/ERK5) as the bait and identified the scaffolding protein 14-3-3beta. To confirm this interaction, the cloned mouse BMK1 gene was expressed in the TNT^® T7 Quick Coupled Transcription/Translation System. The expressed protein was labeled with Transcend™ tRNA.  Using a GST-14-3-3beta fusion protein, a pull-down assay was performed and the direct binding confirmed after immunoblotting and staining with streptavidin-horseradish peroxidase (HRP).  The interaction of various BMK1 mutants were tested in a mammalian two-hybrid system and measured by the Dual-Luciferase^® Reporter Assay System. (3078)

Notes: GoTaq^® DNA Polymerase was used to amplify cDNAs created from total RNA of various yeast strains. Primers specific to ACT1 mRNA and 27S rRNA were used to analyze transcription levels under various conditions.  (3226)

Notes: The authors of this article describe using the SV 96 Total RNA Isolation System on the Beckman-Coulter BioMek^® FX automated laboratory instrument to purify total RNA from 96 separate 2 x 10⁵ HUVEC cell cultures. To demonstrate the quality and consistency of RNA yield, the isolated RNA was reverse transcribed and used in real-time PCR.  (3090)

Notes: Toxoplasma gondii parasites and BS-C-1 cells were incubated for 60 minutes in Hanks' buffered saline solution (HBSS) containing either 100 µM small molecule being screened as potential invasion inhibitors or 0.25% DMSO. Cytotoxicity was determined using the CellTiter-Glo® Luminescent Cell Viability Assay. (3142)

Notes: Several different sequences from a S. pneumoniae pA promoter region were cloned into the pSP-luc+ vector and screened for expression levels in in vitro transcription/translation systems. The RiboMAX™ SP6 Large Scale RNA Production System was used to transcribe luciferase encoding mRNAs.  Luciferase mRNAs and plasmids were used as templates in high throughput inhibition studies in an S. pneumoniae S30 extract described in the paper as well as in Promega’s E. Coli S30 Extract System for Circular DNA and in Rabbit Reticulocyte Lysate. Promega amino acid mixtures were also used in these studies. (3227)

Notes: These authors investigated the possible interaction between the TGFß and PKA signaling pathways. Mv1Lu (fetal mink lung) cells or pancreatic acini from male Swiss Webster mice were treated with TGFß1, washed and protein extracted.  After determining concentration, equal amounts of protein were used in the SignaTECT^® cAMP-Dependent Protein Kinase (PKA) Assay System. The CREB Consensus Oligonucleotide was used in gel shift and supershift assays with nuclear extract prepared from Mv1Lu cells treated with increasing concentrations of TGFß1 to demonstrate the effect of the growth factor on DNA binding.  Cell proliferation was measured using the CellTiter 96^® AQ_ueous Non-Radioactive Cell Proliferation Assay. (3114)

Notes: The research in this article describes differences in the regulatory region of the lactate dehydrogenase-B (Ldh-B ) gene between isolates of Fundulus heteroclitus. To study Ldh-B regulation, 5’ regulatory sequences were cloned from isolates that live at different temperatures. These sequences were subsequently cloned upstream of the firefly luciferase gene. Between 10-60μg of this construct and 5μg of pRL-CMV Vector in physiological saline were injected directly into the liver of the fish using tempera paint as an injection indicator. Seven days after injection, the livers were removed and homogenized with a Polytron homogenizer in Passive Lysis Buffer. The lysate was cleared twice at 10,000 x g for 15 minutes and then stored frozen for 24 hours before being used in the Dual Luciferase^® Assay. (3068)

Notes: HUVECs were infected with adenoviruses encoding either GFP, forkhead transcription factor (FKHR), or FKHR-TM for 24, 32, or 45 hours. At each time point, the relative number of living cells was determined by the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3155)