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Biochem. Pharmacol. 147, 55–66. Characterisation of endogenous A2A and A2B receptor-mediated cyclic AMP responses in HEK 293 cells using the GloSensor™ biosensor: Evidence for an allosteric mechanism of action for the A2B-selective antagonist PSB 603. 2018

Goulding, J., May, L.T. and Hill, S.J.

Notes: HEK293 cells stably expressing the GloSensor™-20F cAMP Plasmid were used to study the molecular pharmacology of the A2BAR -selective antagonist PSB 603. The HEK293 cells endogenously express the human A2BAR receptor and the GloSensor™ system allowed for real-time kinetic analysis of receptor activation by reporting the levels of cellular cAMP following compound treatment. The authors were able to measure both the extent of receptor activation and also the kinetic profile of cAMP levels in the cell and concluded that PSB 603 acts as a negative allosteric modulator of A2BAR -mediated increases in cAMP accumulation. (4970)

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Anal. Chem. 85, 907-14. Mass spectrometry compatible surfactant for optimized in-gel protein digestion. 2013

Saveliev, S.V., Woodroofe, C.C., Sabat, G. Adams, C.M., Klaubert, D., Wood, K., and Urh, M.

Notes: This paper describes use of the mass-spectrometry-compatible ProteaseMax™ Surfactant to improve protein identification for in-gel digestion applications. The surfactant induced a 1.5−2 fold increase in peptide recovery from gel slices, increased sequence coverage 20−30%, and increased the number of identified proteins. The surfactant also accelerated the digestion process. Maximal in-gel digestion was achieved in as little as one hour, depending on incubation temperature, and peptides were readily recovered from the gel, eliminating the need for postdigestion extraction. (4434)

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Mol. Cell. Proteomics 12, 3330–38. Rapid and deep human proteome analysis by single-dimension shotgun proteomics. 2013

Pirmoradian, M., Budamgunta, H., Chingin, K., Zhang, B., Astorga-Wells, J. and Zubarev, R.A.

Notes: These authors evaluated three commonly used cell lysis buffers based on the number of proteins and peptides identified in shotgun UPLC-MS analysis. Detergents used included urea, SDC, and ProteaseMAX™ Surfactant. The proteins were digested with trypsin. The buffer that included ProteaseMax Surfactant gave the greatest number of peptide and protein group identifications. This increase was due to better extraction of membrane, nuclear and cytosolic fractions. (4440)

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J. Biol. Chem. 287, 21599-21614. Proteomic analysis of wild-type and mutant huntingtin-associated proteins in mouse brains identifies unique interactions and involvement in protein synthesis. 2012

Culver, B.P., Savas, J.N., Park, S.K., Choi, J.H., Zheng, S., Zeitlin, S.O., Yates, J.R., and Tanese, N.

Notes: These authors analyzed and compared affinity-purified protein complexes from brain homogenates of wild type and huntingtin (Htt) mutant mice by mass spectrometry. Brain tissue from FLAG-tagged wild-type and Htt mice was homogenized in HEPES buffer supplemented with protease inhibitors and RNasin®. After affinity purification, protein complexes were digested using Sequencing-Grade Modified Trypsin and ProteaseMAX™ Trypsin Enhancer prior to mass-spectrometry analysis. (4227)

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Microbiology 157, 557–65. Differential proteome analysis of Mycobacterium avium subsp. paratuberculosis grown in vitro and isolated from cases of clinical Johne’s disease 2011

Weigoldt, M., Meens, J., Doll, K., Fritsch, I., Möbius, P., Goethe, R. and Gerlack, G.F.

Notes: The authors of this paper were investigating the role of mycobacterial membrane proteins in pathogenesis of Mycobacterium avium subsp. paratuberculosis, causative agent of Bovine Johne’s disease. Membrane enriched protein fractions, either mucosa-derived membranes (MDM) or culture-derived (CDM) of M. avium paratuberculosis from three cows with clinical disease were examined. Initial 2D DIGE and MALDI-TOF-MS analysis was unsatisfactory, so the researchers subjected the membrane preparations to tube-gel trypsin digestion supplemented with the ProteaseMax™ Surfactant, Trypsin Enhancer. They analyzed the proteins using nanoflow-liquid-chromatography-coupled tandem MS. A total of 130 proteins were detected in both MDM and CDM, with 48 predicted membrane proteins; four were not detected in the CDM, implying differential expression in the host. (4214)

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Sci. Signal. 4, ra51. Distinct Phosphorylation Sites on the β2-Adrenergic Receptor Establish a Barcode That Encodes Differential Functions of β-Arrestin 2011

Nobles, K.N., Xiao, K., Ahn, S., Shukla, A.K., Lam, C.M., Rajagopal, S., Strachan, R.T., Huang, T.Y., Bressler, E.A., Hara, M.R., Shenoy, S.K., Gygi, S.P. and Lefkowitz, R.J.

Notes: The authors created a stably transfected HEK293 cell line expressing a luminogenic cAMP-binding protein using GloSensor™ technology to quantify cAMP levels in live cells. The HEK293 cells express a beta2-adrenergic receptor (β2AR ), a Gs-coupled receptor, that when activated with an agonist, stimulates the production of cAMP. The cell line was used to demonstrate that the phosphorylation pattern (“barcode”) of the β2AR created by various G protein-coupled receptor kinases (GPKs) affects the binding and function of beta-arrestin and subsequent internalization of β2AR. GloSensor™ transfection and transcription were confirmed by stimulation of endogenous β2AR with isoproterenol. Endogenous β2ARs in HEK293 cells were prestimulated with either vehicle DMSO or isoproterenol, then washed and rechallenged with serially diluted isoproterenol. In cells transfected with control siRNA, pretreatment with isoproterenol induced a 50% loss of the maximal cAMP signal when rechallenged. Cells transfected with siRNA targeting GRK2, GRK6, or both GRK2 and GRK6 showed impairment of this desensitization. This GRK knockdown effect decreased the change observed in the maximal cAMP response (Emax) after isoproterenol pretreatment. (4138)

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Brain 134, 678–92. Fumaric acid esters exert neuroprotective effects in neuroinflammation via activation of the Nrf2 antioxidant pathway 2011

Linker, R.A., Lee, D-H., Ryan, S., van Dam, A.M., Conrad, R., Bista, P., Zeng, W., Hronowsky, X., Buko, A., Chollate, S., Ellrichmann, G., Brück, Dawson, K., Goelz, S., Wiese, S., Scannevin, R.H., Lukashev, M. and Gold, R.

Notes: The authors of this paper investigated the molecular mechanisms through which fumaric acid esters affect the progression and pathology of multiple sclerosis. The authors looked at the activation of the Nrf2 transcriptional pathway which plays a role in defense against oxidative stress. A stable reporter cell line expressing eight copies of the gluthione-S-transferase 2 antioxidative response elements cloned upstream of the luciferase complementary DNA in a pGL4.26 vector. Reporter cells were stimulated with dimethylfumarate for 24 hours and luciferase activity measured using the Luciferase Assay System. A strong dose-dependent induction of antioxidative response elements was observed. Next, the authors looked at the effects of fumaric acid esters on the inhibitor of Nrf2, Keap 1. C-terminal, V5-tagged Keap1 protein was transfected into 293 cells. Forty-eight hours post transfection, cells were treated with dimethyl fumarate. Samples were lysed, Keap1-V5 was immunoprecipitated and gel purified. The protein was digested with trypsin in the presence of ProteaseMAX™ Surfactant, Trypsin Enhancer. Peptide pools were analyzed using liquid chromatography-tandem mass spectrometry. The authors were able to show that the Keap1 protein was modified in response to treatment. (4215)

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J. Biol. Chem. 286, 24977–86. Mechanism of a genetically encoded dark-to-bright reporter for caspase activity 2011

Nicholls, S.B., Chu, J., Abbruzzese, G., Tremblay, K.D. and Hardy, J.A.

Notes: The authors of this study used GFP to create a fluorescent reporter that can report the functional state of proteins that are activated by a post-translational event such as enzymatic cleavage or other modification. They created a caspase-activatable GFP (CA-GFP) and analyzed the mechanism of action of the reporter. They subjected purified CA-GFP and GFP to trypsin digestion in the presence of ProteaseMax™ Surfactant, Trypsin Enhancer for matrix-assisted-laser desorption Time-of-Flight (MALDI-TOF) and subsequent MS/MS. They were able to show that the absorbance spectra of CA-GFP does not have the characteristic signature of GFP suggesting that the GFP chromaphore is not mature and in the dark state (Figure 6 in the paper). (4216)

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J. Proteome Res. 10, 5150–62. Ookinete-interacting proteins on the microvillar surface are partitioned into detergent resistant membranes of Anopheles gambiae midguts 2011

Parish, L.A., Colquhoun, D.R., Mohien, C.U., Lyashkov, A.E., Graham, D.R. and Dinglasan, R.R.

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)

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J. Proteome Res. 10, 518–28. The effect of silicon on the leaf proteome of rice (Oryza sativa L.) plants under cadmium stress 2011

Nwugo, C.C. and Huerta, A.J.

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)

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Nucl. Acids Res. Feb 17 (Epub), doi:10.1093/nar/gkq087. A cooperative and specific DNA-binding mode of HIV-1 integrase depends on the nature of the metallic cofactor and involves the zinc-containing N-terminal domain. 2010

Carayon, K., Leh, H., Henry, E., Simon, F., Mouscadet, J.F., and Deprez, E.

Notes: The authors of this paper investigated the mechanism of interaction of HIV-1 Integrase with host cell DNA. To understand the role of the integrase Zn-binding domain, they studied the effect of the Zn ejector 2,2'-dithiobisbenzamide (DIBA) on DNA binding. The covalent modification of integrase by DIBA was evaluated using in-gel tryptic digestion and SELDI mass spectrometry analysis. ProteaseMAX Surfactant was used to enhance protein digestion. (4080)

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Mol. Cell. Proteomics 8(4), 827-845. Discovery and scoring of protein interaction subnetworks discriminative of late stage human colon cancer. 2010

Nibbe, R.K., Markowitz, S., Myeroff, L., Ewing, R., and Chance, M.R.

Notes: These authors sought to identify protein signatures associated with late-stage human colorectal cancer. They used 2D gel electrophoresis to compare the protein signatures of normal tissue and tumor samples. Proteins of interest were excised from gels, trypsin digested and analysed by reverse phase LC-MS. The data were compared to existing databases to try to identify specific proteins or protein interactions associated with late-stage tumor tissue. ProteaseMax Surfactant was used to enhance trypsin digestion. (4084)

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Anal. Biochem. 400(1), 61-68. Enrichment of cysteinyl adducts of human serum albumin. 2010

Funk ,W.E., Li, H., Iavarone, A.T., Williams, E.R., Riby, J., and Rappaport, S.M.

Notes: This study describes an enrichment method for cysteinyl adducts of human serum albumin. Electrospray ionization mass spectrometry was used to detect mercaptalbumin and HSA-Cys34 modifications before and after enrichment. After enrichment, mercaptalbumin was no longer observed in mass spectra. Trypsin and ProteaseMax Surfactant were used to prepare samples for mass spectrometry analysis. (4083)

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J. Proteome Res. 9(4), 1882-1893. Identification of a novel family of snake venom proteins Veficolins from Cerberus rynchops using a venom gland transcriptomics and proteomics approach. 2010

OmPraba, G., Chapeaurouge, A., Doley, R,, Devi, K.R., Padmanaban, P., Venkatraman, C., Velmurugan, D., Lin, Q., and Kini, R.M.

Notes: These authors characterized the venom composition of the water snake Cerberus rynchops. As part of the study, whole venom was trypsinized and fractionated by reverse phase HPLC followed by MALDI-MS/MS analysis. For in-solution tryptic digestion, lyophilized crude venom (500 μg) was dissolved in ammonium bicarbonate and precipitated with ice-cold acetone for 3 h at -15 °C. The pellet was resuspended in 1 ml 50 mM ammonium bicarbonate containing 20 μl of 1% ProteaseMAX Surfactant. After reduction with 10 μl 0.5M DTT (at 56 °C for 20 min) and alkylation with 27 μl of 0.55M iodoacetamide (at room temperature for 15 min in the dark), the venom was digested at 37 °C for 3 h using 18 μg trypsin (1 μg/μl) and 10 μl of 1% ProteaseMAX surfactant to improve tryptic cleavage activity. (4081)

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J. Proteome Res. 9(1), 359-372. Post-translational modifications to Toxoplasma gondii alpha- and beta-tubulins include novel C-terminal methylation. 2010

Xiao, H., El Bissati, K., Verdier-Pinard, P., Burd, B., Zhang, H., Kim, K., Fiser, A., Angeletti, R.H., and Weiss, L.M.

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)

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Biochemistry 49(1), 95-102. Protein-protein recognition between acyltransferases and acyl carrier proteins in multimodular polyketide synthases. 2010

Wong F.T., Chen A.Y., Cane D.E., and Khosla C.

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)

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J. Proteome Res. 8(6), 2838-2850. A straightforward and highly efficient precipitation/on-pellet digestion procedure coupled with a long gradient nano-LC separation and Orbitrap mass spectrometry for label-free expression profiling of the swine heart mitochondrial proteome. 2009

Duan, X., Young, R., Straubinger, R.M., Page, B., Cao, J., Wang, H., Yu, H., Canty, J.M., and Qu, J.

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)

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J. Biol. Chem. 284, 21307-21316. High yield heterologous expression of wild-type and mutant Cu+-ATPase (ATP7B, Wilson disease protein) for functional characterization of catalytic activity and serine residues undergoing copper-dependent phosphorylation. 2009

Pilankatta, R., Lewis, D., Adams, C.M., and Inesi, G.

Notes: These authors used proteolysis and mass spectrometry to identify the specific protein domains involved in copper-dependent phosphorylation. They expressed wildtype and mutant ATP7B in COS1 cells. The band of interest was excised from a gel, cut into small pieces and subjected to tryptic digestion in the presence of ProteaseMax Surfactant. (4086)

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Science 322, 1113-1116. Arabidopsis stomatal initiation is controlled by MAPK-mediated regulation of the bHLH SPEECHLESS. 2008

Lampard, G.R., Macalister, C.A., Bergmann, D.C.

Notes: These authors showed that the stomatal initiating factor SPEECHLESSS (SPCH) is a substrate of the kinases MPK3 and MPK6 in vitro, that specific phosphorylation sites on SPCH regulate its activity in vivo, and that components of the stomatal development signaling network modulate SPCH. As part of the study, coomassie-stained gel bands containing the SPCH protein were excised and digested using trypsin and ProteaseMax Surfactant. Mass spectrometry was used to assess phosphorylation at several functionally critical sites. (4087)

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Endocrinology 146, 2749-2759. Astrocyte-derived transforming growth factor-β mediates the neuroprotective effects of 17β-estradiol: Involvement of nonclassical genomic signaling pathways. 2005

Dhandapani, K.M., Wade, F., Mahesh, V.B., and Brann, D

Notes: This study investigated the signaling mechanisms involved in 17β-estradiol- (E2) and tamoxifen-induced release of neurotrophic factors from rat cortical astrocyte cultures. The TGFβ1, TGFβ2, BDNF and GDNF Emax® ImmunoAssay Systems were used to measure the release of these neurotrophic factors from astrocyte cultures treated with E2 or tamoxifen. Both E2 and tamoxifen induced the release of TGFβ1 and TGFβ2, but did not stimulate release of BDNF or GDNF. The PI3K inhibitors LY294002 and wortmanin, and the Akt inhibitor blocked TGFβ release, suggesting the involvement of the PI3K/Akt signaling pathway. The MAPK kinase inhibitors PD98059 and U0126 had no effect. E2 was found to induce transient Akt Ser473 phosphorylation, and this effect was blocked by LY294002 pretreatment, demonstrating a role for PI3K in the observed E2-mediated Akt phosphorylation. (3479)

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J. Neurosci. 25, 11719-11729. Distinct mechanisms for neurotrophin-3-induced acute and long-term synaptic potentiation. 2005

Je, H-S., Zhou, J., Yang, F., and Lu, B.

Notes: This study investigated the mechanisms of acute and long-term synaptic modulation by neurotrophin-3 (NT-3). The investigators used the Xenopus neuromuscular junction synapse as a model system in which to characterize the effects of short- and long-term exposure to NT-3. Three features were found to be required for long-term, but not acute, effects: 1) endocytosis of the NT-3-receptor complex, 2) Akt activation, and, 3) new protein synthesis. As part of the investigation biotinylated NT-3 was supplied to cultures on streptavidin-coated beads, which were too large to be internalized by endocytosis. In this experiment long-term effects were eliminated but acute effects were unchanged, supporting the requirement for endocytosis of NT-3 for long-term effects. To confirm that NT-3 was tightly bound to the beads, the NT-3-coated beads were vortexed for 30 seconds, and the supernatant collected by centrifugation (5 minutes) and assayed for NT-3 content using the NT-3 Emax® ImmunoAssay System. (3501)

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Nat. Neurosci. 7, 596-604. Dynein motors transport activated Trks to promote survival of target-dependent neurons. 2004

Heerssen, H.M., Pazyra, M.F. and Segal, R.A.

Notes: This paper describes an assay to test BDNF dissociation from FluoSpheres (amine-modified microspheres, 1μM diameter; Molecular Probes). BDNF was covalently attached to the microspheres. Release of BDNF was tested in supernatants from the BDNF-microspheres in PBS, in complete media, and in culture with rat Dorsal root ganglia. Promega’s BDNF Emax® ImmunoAssay System was used to determine the amounts of BDNF in these samples. The DeadEnd™ Fluorometric TUNEL System was used to examine rat Dorsal root ganglia cells that had been transfected with either dynamitin or β-Galactosidase. In the TUNEL experiments, cells were fixed in 4% paraformaldehyde and counterstained with DAPI. Cells were also stained with Promega’s Anti-β-Galactosidase, Purified Monoclonal Antibody. For these immunocytochemical stains, a 1:500 dilution of Anti-β-Galactosidase antibody and a 1:1,500 dilution of a secondary antibody conjugated to Alexa Fluor-488 (Molecular Probes) were used. The stains were visualized by fluorescence microscopy. (3055)

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J. Neurosci. 24, 10542-10552. RACK1 and brain-derived neurotrophic factor: a homeostatic pathway that regulates alcohol addiction. 2004

McGough, N.N., He, D.Y., Logrip, M.L., Jeanblanc, J., Phamluong, K., Luong, K., Kharazia, V., Janak, P.H. and Ron D.

Notes: The authors examine the role of brain-derived neurotrophic factor (BDNF) in alcohol addition. RNA was isolated from primary rat hippocampal neurons cultured in the absence or presence of ethanol. The RNA was reverse transcribed using the Reverse Transcription System, and BDNF and GPDH RNAs were quantitated by fluorescent real-time PCR. BDNF and nerve growth factor (NGF) protein levels were monitored using the BNDF and NGF Emax® ImmunoAssay Systems. (3441)

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J. Neurosci. 21, 8789-8797. An activity-dependent neurotrophin-3 autocrine loop regulates the phenotype of developing hippocampal pyramidal neurons before target contact. 2001

Boukhaddaoui, H., Sieso, V., Scamps, F. and Valmier, J.

Notes: Rat embryonic hippocampal neurons were obtained from pregnant Sprague Dawley rats and the NT-3 Emax® ImmunoAssay System was used to assess NT-3 levels. The ELISA protocol was modified as follows: after blocking the plate, neurons were added, also, calcium channel blockers were added after four hours. Thereafter the protocol was as given in the NT-3 Emax® ImmunoAssay System Technical Bulletin. (2806)

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J. Neurochem. 76, 201-9. Neurotrophin-3 mediates the autocrine survival of the catecholaminergic CAD CNS neuronal cell line. 2001

Horton, C.D., Qi, Y., Chikaraishi, D., and Wang J.K.

Notes: A novel catecholaminergic central nervous system cell line capable of autocrine survival mediated by neurotrophin-3 was characterized. This mouse cell line, CAD, can survive and differentiate in the absence of exogenously provided neurotrophic factors. NT-3 levels in CAD  extracts were quantitated using Promega's NT-3 Emax® ImmunoAssay System. (2337)

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