Notes: HeLa cells were grown in 24-well plates and transfected with nontarget and p60 siRNAs with untransfected cells as a control. After 24 and 48 hours, loss of cell viability upon silencing of p60 was assessed by incubating the cells with the CellTiter 96® AQueous One Solution Cell Proliferation Assay for five minutes and then stopping the reaction. (3156)

Notes: Potential compounds to inhibit the SARS-virus-mediated cytopathic effect were dissolved in DMSO to 10 mM. To test for possible cytotoxic effects of the small molecules on the host cells, the Vero E6 cells were incubated with the compounds at varied concentrations for two days and assayed with the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay. The culture medium was replaced with 100 µL MTS phenazine methosulfate in DMEM and incubated at 37°C for 2 hours. (3150)

Notes: Real-time TaqMan^® amplification reactions were cleaned up using the Wizard^® MagneSil^® Sequencing Reaction Clean-Up System.  The purified products were then used in sequencing reactions.  This paper also describes use of the Dual-Luciferase^® Reporter Assay System to analyze HEK293 cells transfected with a pGL3-Enhancer vector construct.  (3181)

Notes: The Beta-Glo® Assay System was used to normalize luciferase readings in dual-reporter transfection studies.  In these experiments, human prostate carcinoma PC-3 cells were transfected with a NF-κB factor-driven luciferase reporter construct and a undefined beta-galactosidase reporter construct.  Cells were treated with 20 and 40µM apigenin for 16 h or with 10ng/ml TNF-α for 30 minutes.  Luciferase reporter enzyme expression was assessed with the Luciferase Assay System.  (3054)

Notes: Mf4/4 cells seeded at 10⁶ cells/well of six-well plates in medium without antibiotics. After 15 hours, the cells were infected with multiplicity of infection of 50 with the Yersinia enterocolitica YopPE- or YopPE- strains that were complemented with wild type (YopEWT) or mutant YopE (YopEM). The cells were analyzed for cytotoxicity after 24 hours using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. (3158)

Notes: The authors found that phosphorylation of a phosphotyrosine binding domain, Fe65, by active c-Abl stimulates amyloid precursor protein/Fe65 transcriptional activity. GAL4UAS-dependent firefly luciferase and transfection-control Renilla luciferase reporter plasmids were created and propagated in CHO and COS-7 cells. The Dual-Glo™ Luciferase Assay System was used to monitor firefly and Renilla luciferase activity, with the firefly activity standardized against the Renilla luciferase control activity. (3539)

Notes: In this study, the psiCHECK-2 vector was used to assist in selection of siRNA sequences that work optimally against their selected target. Dicer and DGCR8 coding sequences were individually cloned into the psiCHECK-2 multiple cloning site. The resulting vector and a synthetic siRNA duplex targeting the gene coding sequence were transfected into 293 cells and  Renilla luciferase activity was measured using the Dual Luciferase^® Assay System. Firefly luciferase activity was used to normalize the data.  siRNAs that caused greater than 80% reduction in Renilla luciferase signal were selected for further use. (3220)

Notes: These authors performed gel shift (EMSA) assays to determine whether purified VirA binds to the vapA promoter. Radiolabeled DNA fragments for the EMSA assays were prepared using DNA Polymerase I Large (Klenow) Fragment. Primer extension using ImProm-II™ Reverse Transcriptase localized the transcription start site within the vapA promoter. To characterize the transcriptional organization of the virR gene cluster, the authors performed reverse transcription using ImProm-II™ Reverse Transcriptase and Random Primers, followed by PCR using a combinations of primers in opposite orientations throughout the gene cluster. The plasmids used in this study were purified by alkaline lysis method or using the Wizard^® Plus SV Minipreps DNA Purification System.
(3563)

Notes: Aliquots of 5 x 10⁴ cells/well were distributed in 96-well plates and incubated with 1, 2, and 5 µM novel synthetic oleanane triterpenoid CDDO for 48 hours. Then the relative cell viability was determined by comparing untreated control (DMSO) to treated groups using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3164)

Notes: Shigella flexneri serotype 5a virulent wild type or mutant strain was used to infect HeLa cultures. After 90 minutes, the level of LDH in the cell culture medium was assayed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay. (3166)

Notes: In this study, siRNA was used to reduce the level of the PU.1 transcription factor.  The siRNA was generated using the T7 RiboMAX™ Express RNAi System with design assistance from the siRNA Target Designer (http://www.promega.com/siRNADesigner/). Annealed siRNA was purified by isopropanol precipitation. Forty-eight hours after transfecting 2.5ug of siRNA into RAW264.7 cells, RNA and protein were isolated from the cells and the siRNA effect was analyzed by RT-PCR and Western blot. (3062)

Notes: 0.5 x 10⁶ cells/ml were seeded in 96-well plates and incubated for 24 or 48 hours with or without recombinant human TNF-α or recombinant human MCP-1. The CellTiter 96® AQueous One Solution Cell Proliferation Assay was added to the cells and incubated for 4 hours before the absorbance was measured. (3157)

Notes: The authors investigate a potential internal ribosome entry site (IRES) in the 5´ untranslated region (UTR) of cellular inhibitor of apoptosis protein 1 (c-IAP1). The c-IAP1 5´ UTR was amplified, cloned into pGEM®-T Vector, sequenced, then inserted into a dicistronic reporter vector between Renilla and firefly luciferase sequences. Using the Dual-Luciferase® Reporter Assay System, IRES activity was evaluated in Rabbit Reticulocyte Lysate and transiently transfected cells. The pSV-β-Galactosidase Control Vector was used as a control for transfection efficiency. Because splicing events were removing part of the Renilla luciferase coding region, the authors chose to use RNA transfection of cells. The ImProm-II™ Reverse Transcription System was used for the reverse transcription step of RT-PCRs to amplify intercistronic regions of the dicistronic RNA to examine mRNA splicing. (3429)

Notes: The TNT^® Coupled Reticulocyte Lysate System was used to in vitro transcribe and translate [³⁵S]methionine-labeled UNR, an RNA binding protein involved in internal ribosome binding. The labeled UNR protein was used in pulldown assays with various RNA binding protein-GST-fusion constructs.  After RNase A treatment of lysates, only poly(A) binding protein (PABP) co-precipitated with UNR. Data are presented as autoradiographs from precipitation studies.  (3248)

Notes: The effects of tamoxifen and vinblastine were assayed on HepG2 and HL-60 cells using a variety of Promega’s cell-based assays. The CellTiter-Glo^® Luminescent Cell Viability Assay, CellTiter 96^® AQueous One Solution Cell Proliferation Assay, and CellTiter-Blue^® Cell Viability Assay were used to test the effects of 0-100µM tamoxifen on HepG2 cell viability after an incubation period ranging from 0-24 hours. Cell membrane integrity was assessed with the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. The Caspase-Glo^® 3/7 Assay was also used to measure Caspase 3/7 activity after 0-100µM tamoxifen treatment. (3189)

Notes: These authors used mice deficient in the isoaspartyl repair enzyme protein carboxyl methyltransferase (PCMT) to examine immune tolerance. Spleen, lymph node, and thymus cells were removed from 4-6-week-old wildtype and PCMT-/- mice. These cells were sonicated and the total protein content determined prior to measuring  isoaspartyl content using a PCMT vapor diffusion assay, the radioactive protocol for the ISOQUANT^® Isoaspartate Detection Kit.  PBS was used as a negative control and the isoaspartyl delta sleep-inducing peptide was used as a positive control. (3119)

Notes: The authors of this paper used the ProteoLink™ In Vitro Expression Cloning System (Human Adult Brain) to screen for proteins that bind p62, a novel Src homology domain binding protein. The researchers expressed the Human Adult Brain library of proteins in the presence of ³⁵S-labeled methionine using the Gold TNT^® SP6 Express 96 System. The labeled proteins were then added to binding assays in the presence of agarose-immobilized p62 UBA (polyubiquitin binding domain).  Eleven proteins were identified and reported in the study.  (2838)

Notes: To test the effect of BMS-378806, a new small molecule inhibitor of HIV-1, a cell fusion assay was developed. Target cells that stably expressed CD4, CXCR4 or CCR5 receptors and carry a responsive luciferase plasmid were prepared.  Effector cells were transiently transfected with HIV coat protein gp160 from various strains of virus, and a plasmid to activate the responsive element promoting luciferase. Therefore, if the cells fuse, luciferase is activated.  To measure this activation, effector cells were plated with target cells in a 1:2 ratio and seeded into 96-well plates at 1.5 x 10⁴ cells/well and incubated with various concentrations of BMS-378806 for 12-24 hours. Luciferase activity was determined with the Steady-Glo^® Reagent. (2737)

Notes: The SV 96 Total RNA Isolation System was used to isolate total RNA from human umbilical vein endothelial (HUVEC) cells, primary keratinocytes (NHEK-adults) and human rheumatoid arthritis synovial fibroblasts (RASF) infected with adenovirus expressing siRNAs targeted at a variety of messages. The isolated RNA was used in real-time SYBR Green RT-PCR reactions to investigate the amount of message present after infection.  For reverse transcriptase reactions, the researchers used 5-100ng of purified total RNA.  Results were normalized to GAPDH message levels. (2752)

Notes: Cells stably transfected with BLV were seeded at a concentration of 500 cells per well into a 96-well plate. After attachment, cells were assayed for growth every 3 days for 2 weeks using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay. (3162)

Notes: This study investigated the effect of CaMKII on store-operated calcium entry (SOCE).  To do this, Xenopus laevis oocytes were depleted of intracellular calcium stores by treating with thapsigargin for 3 hours.  The oocytes were then injected with an RNA encoding  constitutively active CaMKII.  After homogenization in 80mM β-glycerophosphate, 20mM HEPES pH 7.5, 15mM MgCl₂, 1mM DTT, 1mM sodium vanadate, 50mM NaF plus protease inhibitor cocktail, CaMKII activity was assayed using the SignaTECT Calcium/Calmodulin-Dependent Protein Kinase (CaM KII) Assay System. (2750)

Notes: The Apo-ONE^® Homogeneous Caspase-3/7 Assay was used to assay apoptosis in transiently transfected EcR-293 cells with inducible expression of WT, Asp33Ala, p18, or Glu6Ala Bax.  Caspase activity was assessed at 0, 12, 24, and 36 hours post-induction for each protein in the presence or absence of ALLM, an inhibitor of both calpain and cathepsin. The researchers also measured caspase activity with the Apo-ONE^® Homogeneous Caspase-3/7 Assay on A-549, K-562, and U-937 cells in the presence of calpain and cathepsin inhibitors.  (2783)

Notes: Total RNA was isolated from human testes using the RNAgents® Total RNA Isolation System. The isolated RNA was used in reverse transcription reactions to make cDNAs of HLA-linked OR genes. Promoter regions of HLA-linked OR genes were cloned into the pGL3-Basic Vector. The constructs were then transfected into human embryonic kidney (HEK293) and Odora cells for promoter analysis studies. The Bright-Glo™ Luciferase Assay System was used to generate data from the study.  Results were presented as a comparison to pGL3-Control Vector transfectants. (2730)

Notes: The authors examined the putative Pseudomonas aeruginosa homolog to the E. coli small multidrug resistance gene EmrE and its possible role in P. aeruginosa intrinsic drug resistance. Total RNA was isolated from 1-2ml log-phase or overnight stationary-phase P. aeruginosa PAO1 cultures grown in LB using the SV Total RNA Isolation System. After treating the RNA with DNase, 0.1µg RNA was used in the Access RT-PCR System to measure emrE_Pae expression and the constitutive rpsL gene. The two amplification products were then analyzed on a 1.7% agarose gel. (3074)

Notes: In this paper, caspase-3 activity was measured in mouse renal medullary interstitial cells (MMICs) using the Apo-ONE^® Homogeneous Caspase-3/7 Assay. MMICs grown to confluency in 96-well plates were preincubated with 1mM betaine, sorbitol, or inositol before incubation in 600m OsM media for 24 hours. Researchers then added 100μl of Apo-ONE^® Homogeneous Caspase-3/7 Assay Reagent and the plates were shaken at 300rpm for 30 seconds. The plates were then incubated for another hour at room temperature before results were read. Data is presented as the mean of 4 experiments at the emission OD₅₃₈. (2673)