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| 1. |
Groll, M., Schellenberg, B., Bachmann, A.S., Archer, C.R., Huber, R., Powell, T.K., Lindow, S., Kaiser, M. and Duler, R.
(2008)
A plant pathogen virulence factor inhibits the eukaryotic proteasome by a novel mechanism.
Nature
452
,
755-758
.
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| |
Notes:
The authors of this study investigated the mechanism of action of syringolin A (SylA), which is secreted by virulent strains of the plant pathogen Pseudomonas syringae. They show that SylA inhibits all three activities of the proteasome in vitro. They also used the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay to show that SylA inhibits the chymotrypsin-like activity of the proteasome in SK-N-HS neuroblastoma cells.
(0003846) |
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Products: Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay |
| 2. |
Wong, D.J., Nuyten, D.S.A., Regev, A., Lin, M., Adler, A.S., Segal, E., van de Vijver, M.J. and Chang, H.J.
(2008)
Revealing targeted therapy for human cancer by gene module maps
Cancer Res.
68
,
369-378
.
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| |
Notes:
The authors of this study used the gene module map method to identify genotype-specific therapies for human cancers. They studied breast cancer cell lines that expressed the "mitochondria" and "wound signatures". These cell lines, which are associated with tumors that have poor prognosis, were also shown to have a "proteasome signature" and were sensitive to the proteasome inhibitor bortezomib. The authors used the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay to show that genotype-specific killing of "wound signature" cells was not due to differential degrees of proteasome inhibition, since 1 and 1µmol/L of bortezomib completely inhibited the ability of the wound signature and control cells to cleave the luminogenic substrate. More likely, the wound signature genotype conferred increased susceptibility to death from proteasome inhibition.
(0003870) |
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Products: Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay |
| 3. |
Betzenhauser, M.J., Wagner, L.E. 2nd, Iwai, M., Michikawa, T., Mikoshiba, K. and Yule, D.I.
(2008)
ATP modulation of Ca2+ release by type-2 and type-3 inositol (1, 4, 5)-triphosphate receptors. Differing ATP sensitivities and molecular determinants of action.
J. Biol. Chem.
283
,
21579–87
.
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| |
Notes:
The authors examined the different ATP sensitivities of inositol (1,4,5)-triphosphate receptor (InsP3R) isoforms InsP3R1, InsP3R2 and InsP3R3. To compare the ATP-binding properties of InsP3R2 and InsP3R3, nucleotide sequences encompassing the ATP-binding domains were amplified by PCR and cloned into the pFN2A (GST) Flexi® Vector. The ATP-binding sites were expressed as glutathione-S-transferase (GST) fusion proteins in BL21(DE3)pLysS cells. Fusion proteins were purified, and the GST tag removed by cleavage with tobacco etch virus (TEV) protease. Purified proteins were then used in a fluorescent ATP-binding assay.
(0003901) |
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Products: BL21(DE3)pLysS Competent Cells, >106cfu/μg | pFN2A (GST) Flexi® Vector |
| 4. |
Frederick, R.O., Bergeman, L., Blommel, P.G., Bailey, L.J., McCoy, J.G., Song, J., Meske, L., Bingman, C.A., Riters, M., Dillon, N.A., Kunert, J., Yoon, J.W., Lim, A., Cassidy, M., Bunge, J., Aceti, D.J., Primm, J.G., Markley, J.L., Phillips, G.N. Jr. and Fox, B.G.
(2008)
Small-scale, semi-automated purification of eukaryotic proteins for structure determination.
J. Struct. Funct. Genomics
8
,
153-166
.
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Notes:
These authors describe a simple, small-scale screening method for recombinant polyhistidine-tagged proteins. They used the Maxwell® 16 Polyhistidine Protein Purification Kit and Maxwell® 16 Instrument to purify the proteins, and characterized the purified proteins by NMR and X-Ray analysis. They first used the Flexi® Vector System to clone the genes of interest into expression vectors containing either an N-terminal TEV protease cleavable His8-Maltose Binding Protein (His8-MBP) tag, or an in vivo cleaved His8-MBP tag. For small-scale screening, E. coli expressing fusion proteins were grown for 24 hours in auto-induction medium in 96-well growth blocks, harvested by centrifugation and resuspended to an OD600 of 20 in 1ml of 50mM HEPES (pH 7.5) containing a protease inhibitor cocktail. Aliquots of these cells were then added directly to the Maxwell® kit cartridge, and automated purification performed. The purified proteins were analyzed by SDS-PAGE or using a Caliper LC90 electrophoresis system. For purification of proteins for use in NMR or crystallography studies, 50ml overnight cultures were harvested and resuspended in 10 ml of 50mM HEPES (pH 7.5) containing 10 units of benzonase, 1mg/ml lysozyme, and protease inhibitor cocktail. The cell suspension was sonicated, and aliquots were then added to the Maxwell® 16 cartridge for purification. The authors purified 14 different proteins from humans, frogs, and zebrafish using this method. Detailed reports of yields obtained and subsequent analyses are provided in the paper.
(0003799) |
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Products: Maxwell® 16 Instrument | Maxwell® 16 Polyhistidine Protein Purification Kit |
| 5. |
Cali, J.J., Niles, A., Valley, M.P., O’Brien, M.A., Riss, T.L., and Shultz, J.
(2008)
Bioluminescent assays for ADMET
Expert Opin. Drug Metab. Toxicol.
4
,
103–120
.
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| |
Notes:
The authors of this review article highlight the use of bioluminescence as a readout for high-throughput ADME/Tox assays. They discuss three strategies for designing bioluminescent assays, using either luciferase, ATP or luciferin substrates as the limiting reagents for a luciferase-catalyzed reaction. Reporter gene assays limit the production of luciferase by tying it to a promoter or DNA regulatory region of interest. Such assays can be used to study genes that are regulated by drugs and other xenobiotics. Bioluminescent assays in which ATP is the limiting reagent of the luciferase reaction can be designed to monitor cell viability or the activity kinases. Bioluminescent assays in which the substrate is limiting can be designed so that the activity of a particular enzyme results in the production of a luciferin substrate that can, in turn, be acted upon by luciferase.
(0003926) |
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Products: Apo-ONE® Homogeneous Caspase-3/7 Assay | Apo-ONE® Homogeneous Caspase-3/7 Buffer | Beta-Glo® Assay System | Calpain-Glo™ Protease Assay | Caspase-Glo® 2 Assay | Caspase-Glo® 3/7 Assay | Caspase-Glo® 6 Assay | Caspase-Glo® 8 Assay | Caspase-Glo® 9 Assay | CellTiter-Glo® Luminescent Cell Viability Assay | GloResponse™ CRE-luc2P HEK293 Cell Line | GloResponse™ NFAT-RE-luc2P HEK293 Cell Line | GSH-Glo™ Glutathione Assay | Kinase-Glo® Luminescent Kinase Assay | Kinase-Glo® Max Luminescent Kinase Assay | Kinas |
| 6. |
Kojima, K., Tsuzuki, S., Fushiki, T. and Inouye, K.
(2008)
Roles of functional and structural domains of hepatocyte growth factor activator inhibitor type 1 in the inhibition of matriptase.
J. Biol. Chem.
283
,
2478–2487
.
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Notes:
The authors determined the role of various domains of the hepatocyte growth factor activator inhibitor type 1 (HAI-1) in the inhibition of the protease matriptase. HAI-1 mutants lacking one or more domains were expressed as His-tagged fusion proteins in CHO-K1 or COS-1 cells, and proteins were purified using the HisLink™ Protein Purification Resin. Purified proteins were then used in protease assays with recombinant matriptase.
(0003788) |
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Products: HisLink™ Protein Purification Resin |
| 7. |
Seyb, K.I., Schuman, E.R., Ni, J., Huang, M.M., Michaelis, M.L., and Glicksman, M.A.
(2008)
Identification of small molecule inhibitors of β-amyloid cytotoxicity through a cell-based high-throughput sreening platform.
J. Biomol. Screening
13
,
870-878
.
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| |
Notes:
This paper demonstrates use of a calpain assay in a cell-based format. (Calpain-Glo™ Assay).
(0003941) |
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Products: Calpain-Glo™ Protease Assay |
| 8. |
Pilecka, I., Patrignani, C., Pescini, R., Curchod, M.L., Perrin, D., Xue, Y., Yasenchak, J., Clark, A., Magnone, M.C., Zaratin, P., Valenzuela, D., Rommel, C. and van Huijsduijnen, R.H.
(2007)
Protein-tyrosine phosphatase H1 controls growth hormone receptor signaling and systemic growth.
J. Biol. Chem.
282
,
35405-35415
.
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| |
Notes:
To genotype PTPH1 knock-out (KO), heterozygous (HET), and wild type (WT) mice, tail snips were digested overnight with proteinase K and the DNA trapped using the Wizard® SV 96 Genomic DNA Purification System. Genomic DNA was washed using the Wizard® SV Wash Solution and eluted in 200μl of water at 65°C. The protease was inactivated at 95°C and 2μl of DNA was used for PCR.
(0003739) |
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Products: Wizard® SV 96 Genomic DNA Purification System |
| 9. |
Filimonenko, M., Stuffers, S., Railborg, C., Yamamoto, A., Malerod, L., Fisher, E.M.C., Isaacs, A., Brech, A., Stenmark, H. and Simonsen, A.
(2007)
Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease.
J. Cell Biol.
179
,
485-500
.
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| |
Notes:
Endosomal sorting complexes required for transport (ESCRTs) are necessary for sorting membrane proteins into the intralumenal vesicles of the multivesicular body for eventual degradation by the lysosome/vacuole. Mutations in at least one subunit of the ESCRTs are associated with frontotemporal dementia and ALS. In this study, the authors demonstrate that ESCRTs are required for autophagy and prevention of protein aggregation. They address the question of whether loss of ESCRTs might interfere with proteasome activity. Using the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay, they show that proteasome activity is minimally affected in ESCRT-depleted cells.
(0003847) |
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Products: Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay |
| 10. |
Huang, L., Ho, P. and Chen, C-H.
(2007)
Activation and inhibition of the proteasome by betulinic acid and its derivatives
FEBS Lett.
581
,
4955-4959
.
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Notes:
The authors of this study investigated the effects of betulinic acid (BA) and its chemical derivatives on the proteasome in vitro and in cells. They used the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay to assess the effects of BA derivatives on proteasome activity in MT4 cells.
(0003871) |
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Products: Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay |
| 11. |
Tanaka, M., Chock, P.B., and Stadtman, E.R.
(2007)
Oxidized messenger RNA induces translation errors.
Proc. Natl. Acad. Sci. U S A
104
,
66-71
.
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| |
Notes:
Oxidative damage has been associated with a range of age-related neurological conditions. In this study, the effect of mRNA oxidation was investigated. A direct correlation was observed between the extent of oxidation and the frequency of translation errors. The authors excised the firefly luciferase (luc2) gene from the pGL4.14 Vector, attached a FLAG tag to the 5´ terminus and a Myc tag to the 3´ terminus, and subcloned the gene into a pGEM-4Z Vector that had been modified to append a poly(A) sequence. The construct was transfected into HEK293 cells, which were then cultured in the presence of an oxidizing agent. The occurrence of truncated protein fragments and short peptides increased in the presence of the oxidizing agent in a concentration-dependent manner. The effects of oxidation of mRNA were also investigated in in vitro translation experiments using mRNA treated with an iron-ascorbate mixture and hydrogen peroxide. Translation in vitro was performed using rabbit reticulocyte lysate supplemented with protease inhibitors. The translation products were detected using anti-FLAG and anti-c-Myc antibodies.
(0003630) |
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Products: pGEM®-4Z Vector | pGL4.14[luc2/Hygro] Vector | Rabbit Reticulocyte Lysate System, Nuclease Treated |
| 12. |
Igarashi, M., Yogiashi, Y., Mihara, M., Takada, I., Kitagawa, H. and Kato, S.
(2007)
Vitamin K induces osteoblast differentiation through pregnane X receptor-mediated transcriptional control of the Msx2 gene.
Mol. Cell. Biol.
27
,
7947-7954
.
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| |
Notes:
Igarashi et al. showed that expression of the Msx2 gene, which enodes a transcription factor, is induced by vitamin K treatment via a pregnane X receptor response element (PXRE) and by estrogen via an estrogen response element (ERE). Promoter analysis was performed by cloning the Msx2 promoters into the pGL3-Basic Vector, transfecting MC3T3 and ST2 cells with the pGL3-Basic constructs, treating the cell with 10nM 17β-estradiol and 10µM vitamin K, then measuring luciferase activity. The pRL-CMV Vector (2.5ng per well of a 12-well plate) was cotransfected to normalize for transfection efficiency. The ability of PXR to bind to the Msx2-PXRE was assessed by an avidin-biotin complex DNA assay. Sense and antisense oligonucleotides that were biotinylated at the 5´ end were annealed and immobilized with the TetraLink™ Tetrameric Avidin Resin. HEK293T cells were lysed with lysis buffer (10mM Tris-HCl [pH7.8], 1mM EDTA, 150mM NaCl, 0.1% NP-40) containing protease inhibitors, then centrifuged to clarify the extract. The supernatants were mixed with the DNA-TetraLink™ Resin to allow proteins to bind to the oligos, and resin was washed with lysis buffer. Bound proteins were analyzed by SDS-PAGE and Western blotting.
(0003805) |
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Products: pGL3-Basic Vector | pRL-CMV Vector | TetraLink™ Tetrameric Avidin Resin |
| 13. |
Niles, A.L., Moravec, R.A., Hesselberth, P.E., Scurria, M.A., Daily, W.J. and Riss, T.L.
(2007)
A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers
Anal. Biochem.
366
,
197–206
.
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| |
Notes:
The authors of this paper describe an assay that uses protease biomarkers to assess cell viability and cell death simultaneously in a population of cells. The assay detects an ubiquitous protease activity that is associated with live cells and a second protease activity that is associated with cells that have lost membrane integrity. The readouts are either fluorescent or fluorescent and luminescent. The assay can be performed in multiplex with other assays, such as caspase assays, to gain additional information on the cell population, and it is amenable to high-throughput screening.
(0003927) |
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Products: CytoTox-Glo™ Cytotoxicity Assay | MultiTox-Fluor Multiplex Cytotoxicity Assay | MultiTox-Glo Multiplex Cytotoxicity Assay |
| 14. |
Fan, F. and Wood, K.V.
(2007)
Bioluminescent assays for high-throughput screening
Assay Drug Dev. Technol.
5
,
127–136
.
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| |
Notes:
The authors of this paper review bioluminescent assay technologies, discussing HTS reporter, cell-based and luciferase biosensor assays. They divide luminescent assays into three basic categories: assays that measure ATP concentration (cell viability and kinase assays), assays that measure changes in luciferase levels (reporter assays, GPCR assays), and assays that measure changes in luciferin levels (protease [including caspase], P450 and MAO assays).
(0003737) |
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Products: Renilla Luciferase Assay System | BacTiter-Glo™ Microbial Cell Viability Assay | Bright-Glo™ Luciferase Assay System | Calpain-Glo™ Protease Assay | cAMP-Glo™ Assay | Caspase-Glo® 2 Assay | Caspase-Glo® 3/7 Assay | Caspase-Glo® 6 Assay | Caspase-Glo® 8 Assay | Caspase-Glo® 9 Assay | CellTiter-Glo® Luminescent Cell Viability Assay | Dual-Glo® Luciferase Assay System | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | Kinase-Glo® Lumine |
| 15. |
Koh Y, Matsumi S, Das D, Amano M, Davis DA, Li J, Leschenko S, Baldridge A, Shioda T, Yarchoan R, Ghosh AK, Mitsuya H.
(2007)
Potent inhibition of HIV-1 replication by novel non-peptidyl small molecule inhibitors of protease dimerization.
J. Biol. Chem.
282
,
28709-28720
.
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Notes:
Dimerization of HIV-1 protease subunuts is essential for proteolytic activity and viral replication. These authors used a FRET assay to identify potential small molecule inhibitors of HIV-1 protease dimerization. After identifying a number of inhibitors in the FRET assay, they used the CheckMate™ Mammalian Two-Hybrid System to independently confirm the disruption of interaction between the two protein subunits.
(0003711) |
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Products: CheckMate™ Mammalian Two-Hybrid System |
| 16. |
Boehmerle, W., Zhang, K., Sivula, M., Heidrich, F.M., Lee, Y. Jordt, S-E., and Ehrlich, B.E.
(2007)
Chronic exposure to paclitaxel diminishes phosphoinositide signaling by calpain-mediated neuronal calcium sensor-1 degradation.
Proc. Natl. Acad. Sci. U S A
104
,
11103-11108
.
|
| |
Notes:
Taxol-induced peripheral neuropathy is a common side-effect of treatment that has been associated with disturbed intracellular calcium homeostasis in neuronal cells. These authors investigated whether prolonged exposure to Taxol caused alterations in calcium signaling in human neuroblastoma and rat dorsal root ganglia. They found that expression of the inositol 1,4,5-triphosphate receptor modulator NCS-1 was reduced in Taxol-treated neuronal cells. The authors also found that Taxol treatment activated calpain, which degrades NCS-1, and that the calpain inhibitor AK295 prevented Taxol-mediated suppression of calcium release. These findings suggest that calcium-mediated activation of calpain is responsible for the observed degradation of NCS-1.
(0003681) |
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Products: Calpain-Glo™ Protease Assay |
| 17. |
Ma, H., Horiuchi, K.Y., Wang, Y., Kucharewicz, S.A. and Diamond, S.L.
(2005)
Nanoliter homogenous ultra-high throughput screening microarray for lead discoveries and IC50 profiling.
Assay Drug Dev. Technol.
3
,
177-87
.
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| |
Notes:
These authors used a microarray format for ultra-high-throughput screening of compounds to determine IC50 values for various biologically relevant enzymes. Assays for protein kinase A (PKA) were performed in nanoliter-volume reactions using the ProFluor® PKA Assay. Chemical compounds (7 or 100µM) from a small library and 5 or 0.75µM PKA substrate were arrayed and activated by aerosol deposition with a mixture of 7 units/µl PKA and 70µM ATP on the DiscoveryDot platform. The chips were then activated again with 5X protease solution. For fluorescence detection and analysis, the chips were scanned, the image files analyzed with the ArrayPro program, and the activity data normalized and plotted with GraphPad Prism® to derive the IC50 values. It was demonstrated that the ProFluor® PKA Assay was amenable to this microarray format (Z' factor >0.6).
(0003283) |
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Products: ProFluor® PKA Assay |
| 18. |
Notebaert, S., Duchateau, L. and Meyer, E.
(2005)
NF-κB inhibition accelerates apoptosis of bovine neutrophils.
Vet. Res.
36
,
229–240
.
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| |
Notes:
The Caspase-Glo™ Assay was used to monitor apoptosis in bovine neutrophils. In these experiments, the researchers slightly modified the Caspase-Glo™ add-and-read procedure. One million neutorphils were treated with a combination of TNF-α and/or gliotoxin for 6 hours before being lysed in a solution of PBS supplemented with protease inhibitors and 1% saponin. The lysates were cleared, stored at -80°C, and then diluted before being analyzed with the Caspase-Glo™ Assay System. Data was presented as the relative light units for each sample.
(0003305) |
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Products: Caspase-Glo® 3/7 Assay |
| 19. |
de Haan, C.A.M., Stadler, K., Godeke, G.J., Bosch, B.J. and Rottier, P.J.M.
(2004)
Cleavage inhibition of the murine coronavirus spike protein by a furin-like enzyme affects cell-cell but not virus-cell fusion.
J. Virol.
78 (11)
,
6048–6054
.
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Notes:
The Renilla Assay System was used to analyze mouse hepatitis coronavirus strain A59 (MHV-A59) entry in to cells. A mouse hepatitis coronavirus construct expressing Renilla luciferase was used to infect LR7 cells in the presence or absence of a Furin protease inhibitor.
(0003041) |
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Products: Renilla Luciferase Assay System |
| 20. |
Mulugeta, S. and Beers, M.F.
(2003)
Processing of surfactant protein C requires a type II transmembrane topology directed by juxtamembrane positively charged residues.
J. Biol. Chem.
278(48)
,
47979-47986
.
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Notes:
These authors showed that double-substitution mutation of two N-terminal juxtaposed residues (from positive to neutral charged species) resulted in a reversal of the transmembrane orientation of the protein of interest. For in vitro transcription/translation, they used the TNT® T7 Coupled Reticulocyte Lysate System in the presence of Canine Pancreatic Microsomal Membranes. A protease protection assay and an epitope-specific pull-down assay were used to determine the membrane orientation of the in vitro synthesized protein.
(0003051) |
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Products: Canine Pancreatic Microsomal Membranes | TNT® T7 Coupled Reticulocyte Lysate System | TNT® T7 Coupled Reticulocyte Lysate System, Trial Size |
| 21. |
Machaca, K.
(2003)
Ca2+-calmodulin-dependent protein kinase II potentiates store-operated Ca2+ current.
J. Biol. Chem.
278(36)
,
33730-33737
.
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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.
(0002750) |
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Products: SignaTECT® Calcium/Calmodulin-Dependent Protein Kinase (CaM KII) Assay System |
| 22. |
Iborra, F.J., Jackson, D.A. and Cook, P.R.
(2001)
Coupled transcription and translation within nuclei of mammalian cells.
Science
293(5532)
,
1139-1142
.
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Notes:
To understand if protein translation occurs in the nucleus of eukaryotic cells, the researchers in this study labeled proteins in vivo by bathing permeablized cells in a translation mix containing radiochemical, biotin or fluorescently tagged amino acids. The study found that all three types of label gave similar results. To fluorescently tag proteins, cells were grown, then permeablized, and bathed in a mix of buffer, creatine phosphokinase, phosphocreatine, GTP, tRNA, aminoacyl-tRNA synthetase, MgCl2, protease inhibitors, a 1:10 dilution of Fluorotect Greenlys BODIPY labeled lysine-tRNA complex and 50μM amino acid mixture minus lysine. The study revealed that, contrary to previous beliefs, some proteins are transcribed and translated within the nucleus in a coupled manner.
(0002744) |
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Products: FluoroTect™ GreenLys in vitro Translation Labeling System |
| 23. |
Leitlein, J., Aulwurm, S., Waltereit, R., Naumann, U., Wagenknecht, B., Garten, W., Weller, M., and Platten, M.
(2001)
Processing of immunosuppressive pro-TGF-beta 1,2 by human glioblastoma cells involves cytoplasmic and secreted furin-like proteases.
J. Immunol.
166
,
7238-7243
.
|
| |
Notes:
Inhibitors of furin-like protease activity interfere with processing and secretion of TGFβ1 and β2 from cells. Levels of TGFβ1 released into spent culture medium of SV40 large T-Ag immortalized fetal human astrocytic cell line Sv-FHAS and the human glioma cell lines LN-18 and T98G were determined by Western blot analysis using the Anti-TGFβ1 pAb (1:2000 dilution). Due to the low protein concentrations, the spent medium was concentrated with the Centriplus centrifugal filter device YM-3 (Millipore) prior to SDS-PAGE.
(0002455) |
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Products: Anti-TGFβ1 pAb |
| 24. |
Xie, Y., and Varshavsky, A.
(2001)
RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: a negative feedback circuit.
Proc. Natl. Acad. Sci. U S A
98
,
3056-61
.
|
| |
Notes:
The RPN4 protein is involved in the regulation of levels of proteasomal subunits in Saccharomyces cerevisiae. The protein was found to be short-lived by fusing the RPN4 degradation signal to the long-lived β-galactosidase protein. Levels of this fusion protein were quantitated by immunoprecipitation of equal amounts of TCA-insoluble 35S labeled protein using the Anti-β-Galactosidase mAb. Yeast cells were lysed by vortexing with glass beads in lysis buffer (1% Triton® X-100/0.15 M NaCl/1 mM EDTA/50 mM Na-Hepes, pH 7.5) containing protease inhibitors prior to immunoprecipitation.
(0002446) |
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Products: Anti-β-Galactosidase, Purified Monoclonal Antibody |
| 25. |
Sperl, S., Jacob, U., de Prada, N.A., Sturzebecher, J., Wilhelm, O.G., Bode, W., Magdolen, V., Huber, R., Moroder, L.
(2000)
(4-Aminomethyl)phenylguanidine derivatives as nonpeptidic highly selective inhibitors of human urokinase
Proc. Natl. Acad. Sci. U S A
97
,
5113-5118
.
|
| |
Notes:
The authors synthesized a new class of nonpeptidic, reversible inhibitors of the serine protease urokinase-type plasminogen activator (uPA). One of these inhibitors was tested for its cytotoxicity against several human carcinoma cell lines (OV-MZ-6, MDA-MB-231, and A431) using the CellTiter 96® Non-Radioactive Cell Proliferation Assay.
(0002476) |
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Products: CellTiter 96® Non-Radioactive Cell Proliferation Assay |