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Search results for: Caspase 6 Substrate VEID pNA200 assays

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#22140457   2011/11/29 To Up

A quantitative method for the specific assessment of caspase-6 activity in cell culture.

Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses of neurodegenerative disorders such as Alzheimer's and Huntington disease. Commercially available assays to measure caspase-6 activity commonly use the VEID peptide as a substrate. However these methods are not well suited to specifically assess caspase-6 activity in the presence of other, confounding protease activities, as often encountered in cell and tissue samples. Here we report the development of a method that overcomes this limitation by using a protein substrate, lamin A, which is highly specific for caspase-6 cleavage at amino acid 230. Using a neo-epitope antibody against cleaved lamin A, we developed an electrochemiluminescence-based ELISA assay that is suitable to specifically detect and quantify caspase-6 activity in highly apoptotic cell extracts. The method is more sensitive than VEID-based assays and can be adapted to a high-content imaging platform for high-throughput screening. This method should be useful to screen for and characterize caspase-6 inhibitor compounds and other interventions to decrease intracellular caspase-6 activity for applications in neurodegenerative disorders.
Dagmar E Ehrnhoefer, Niels H Skotte, Jane Savill, Yen T N Nguyen, Safia Ladha, Li-Ping Cao, Edie Dullaghan, Michael R Hayden

2039 related Products with: A quantitative method for the specific assessment of caspase-6 activity in cell culture.

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#20405221   // To Up

The execution phase of autophagy associated PCD during insect metamorphosis.

During metamorphosis of Manduca sexta, involution of labial glands follows an autophagic pathway towards programmed cell death (PCD). We looked for evidence of both caspase dependent and independent pathways of PCD by assaying for caspases -1, -2, -3, and -6, proteasomal protease, and cathepsins B & L, using fluorogenic substrates and aldehyde and chloromethylketone inhibitors. The substrates FR-AMC and RR-AMC, preferentially degraded by cathepsins B and L, were the most rapidly degraded, increasing in rate as the gland involuted. Digestion of YVAD-AMC (preferential substrate for caspase-1) and DEVD-AMC (substrate for caspases-3 & -7) was barely detectable, less than 0.02% (on a per-unit-protein basis) of that seen in vertebrate embryos induced to undergo apoptosis. Cleavage of VDVAD-AFC (substrate for caspase -2) and VEID-AFC (substrate for caspase -6) was also assessed, but activity was negligible. Mitochondrial membrane permeabilization (MMP) and cytochrome c release were not detected. Exogenous caspase substrate, polyadenosyl ribose phosphorylase (PARP), is cleaved by labial gland extracts, but only at an acidic pH of 5.5-6.0, and into fragments different from those generated by caspases (confirmed by N-terminal sequencing). The cysteine protease inhibitor leupeptin inhibits PARP cleavage, but the caspase inhibitor DEVD-CHO does not. However, potential caspase-derived fragments of PARP are seen when cytochrome c and dATP are added to cytosolic extracts. Although apoptotic machinery is conserved and functional in this tissue, cell death occurs independently of caspases in metamorphosis. We also postulate that lysosomal proteases play the major proteolytic role similar to the caspase cascade seen in apoptosis.
Caroline O B Facey, Richard A Lockshin

2812 related Products with: The execution phase of autophagy associated PCD during insect metamorphosis.

5500 Units10 ug1x10e7 cells10100 IU96tests

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#16683263   // To Up

Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe.

A number of fluorescent caspase substrates and FRET-based indicators have been developed to study the in vivo activation of caspases, a conserved family of proteases critical in inflammatory, and apoptosis signaling pathways. To date, all substrates have measured only one caspase activity. Here, we describe a FRET-based probe for simultaneously measuring two distinct caspase activities in living cells.
Xiaoli Wu, James Simone, Derek Hewgill, Richard Siegel, Peter E Lipsky, Liusheng He

1467 related Products with: Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe.

100 ml.25 ml.96 tests100 20 3 mg20 100

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#11490368   // To Up

A tripartite anoikis-like mechanism causes early isolated islet apoptosis.

This study examines the mechanisms of early isolated islet apoptosis (II-APO) and loss of functional islet mass.
F Thomas, J Wu, J L Contreras, C Smyth, G Bilbao, J He, J Thomas

1667 related Products with: A tripartite anoikis-like mechanism causes early isolated islet apoptosis.

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