Choose an application

• Reference Manager
• EndNote
• RefWorks (Direct export to RefWorks)

Western immunoblotting --- Immunoblotting, Western --- Protein blotting --- Western blot analysis --- Western blotting --- Immunoblotting --- Proteins --- Analysis --- Immunologia --- Proteïnes

Choose an application

• Reference Manager
• EndNote
• RefWorks (Direct export to RefWorks)

Nrf2 (nuclear factor erythroid 2-related factor 2) is a transcription factor that regulates the expression of many antioxidant and cytoprotective genes. In the absence of stress, Nrf2 is bound in the cytoplasm by its repressor, namely Keap1 (Kelch-like ECH-associated protein 1), which is an adapter for the Cul3-based E3-ubiquitin ligase complex. As a consequence, Nrf2 is constantly ubiquitinated and degraded by the proteasome. In condition of oxidative or electrophilic stress, different mechanisms can provoke the liberation of Nrf2 from Keap1, leading to its translocation to the nucleus. There, Nrf2 activates the transcription of its target genes, which all contain a recognition sequence know as the antioxidant response element (ARE) in their promoter region. Nrf2 is a cytoprotective transcription factor that protects healthy cells from the toxicity of various toxic compounds. However, a growing body of evidence suggests that it can also induce a chemoresistance in cancer cells. The goal of our study is therefore to understand how Nrf2 can mediate a chemoresistance in cancer cells. For that purpose, we use MCF-7 breast cancer cells, and tried to actvate Nrf2 by silencing its repressors (cullin-3 and Keap1) with siRNAs Western blot analysis confirmed the inhibition of these proteins, the subsequent activation of Nfr2 and the upregulation of NAD(P)H quinine oxidoreductase (NQO1), a well-known Nfr2 target gene. In addition, we observed that glutathione levels were higher in cells for which Nrf2 was activated, as compared to control cells. We then incubated wells with two chemotherapeutic agents (doxorubicin or mitoxantrone), in the presence or the absence of siRNAs against cullin-3 or Keap1. We observed that cullin-3 or Keap1 silenced cells were more resistant to chemotherapy than control cells. We then performed microarray experiments to determine which genes are actually induced by Nrf2. Finally, we used MCF-7 cells stably transfected which genes are actually induced by Nrf2. Finally, we used MCF-7 cells stably transfected with an (ARE)-luciferase reporter system to assess the ability of different chemotherapeutic agents to activate Nrf2. Our results show that carmustin and chlorambucil, two alkylating agents, are inducers of Nrf2. Taken together, our results point our that Nrf2 can be an important factor determining cancer cell sensitivity to chemotherapy. Further experiments will be required to undertand the precise mechanisms by which Nrf2 exert its cytoprotective effect in cancer cells

NF-E2-Related Factor 2 --- Transcription Factors --- Blotting, Western

Choose an application

• Reference Manager
• EndNote
• RefWorks (Direct export to RefWorks)

Alzheimer’s disease is a neurodegenerative dementia characterized by the presence of neurofibrillary tangles and senile plaques in the brain.
The intraneuronale neurofibrillary tangles are composed of paired helical filaments (PHF) containing the microtubule associated protein TAU.
Senile plaques are extracellular lesions containing an amyloid core surrounded by dystrophic neuritis and glial cells. The major constituent of the amyloid core is a 39-43 amino acid peptide, Aβ, which is derived from a transmembrane protein, the amyloid precursor protein (APP).
In this work, we have analyzed the effects induced by the expression of different forms of human APP in rat cultured neurons.
The adenoviral expression of APP695 in rat cortical neurons induces neurotoxicity. Neuronal death is not triggered by soluble APP or extracellular Aβ.
Expression of the C-terminal fragment of APP, or APP deleted in its C-terminal domain in neurotoxic. Those different APP isoforms accumulate similar levels of intraneuronale Aβ. Treatment of neurons with a specific γ-secretase inhibitor decreases intracellular Aβ accumulation and allows recovery of neuronal survival. These results demonstrate that intraneuronale Aβ accumulation is responsible for the neurotoxicity observed in our model.
To better characterize the observed neurotoxicity, we measured the activity of caspase 3, a maker of apoptosis. Expression of APP in neurons induces caspase 3 activity as compared to untreated neurons. This suggests that intraneuronale Aβ accumulation activated caspase 3.
Surprisingly, treatment of neurons expressing human APP695 with a specific caspase 3 inhibitor has not effect on neuronal survival. Therefore, caspase 3 doesn’t seem to be the only effector responsible for neuronal death observed in our model La maladie d’Alzheimer est une démence dégénérative caractérisée par la présence, dans le tissus cérébral, de deux types de lésions : les dégénérescences neurofibrillaires et les plaques séniles.
Les dégénérescences neurofibrillaires intraneuronales sont composées de paires hélicoïdales de filaments (PHF) dont le constituant majeur est une protéine associée aux microtubes : la protéine TAU.
Les plaques séniles sont des lésions extracellulaires constituées d’un noyau de fibres amyloïdes entouré de neurites dystrophiques et de cellules gliales. Le constituant majeur du noyau amyloïde est un peptide de 39 à 42 acides aminés appelé peptide amyloïde β ou Aβ. Il résulte d’un clivage protéolytique d’un précurseur transmembranaire : le précurseur du peptide amyloïde (APP). Ce peptide Aβ s’accumule également dans les neurones de patients atteints de la maladie d’Alzheimer.
Au cours de ce travail, nous avons analysés, les effets induits par l’expression de l’APP humain dans des neurones de rat en culture.
L’expression d’APP humain des neurones corticaux de rat à l’aide d’adénovirus provoque une neurotoxicité importante. La production d’Aβ-40 et d’APP soluble extracellulaires ne sont pas responsables de la mort neuronale observée.
L’expression du fragment C-terminal de l’APP (C99) ou encore de l’APP délaité de son domaine C-terminal intracellulaire induisent une neurotoxicité comparable à celle de l’APP. L’expression de ces trois formes d’APP provoque l’accumulation intraneuronale par un inhibiteur spécifique de la γ-sécrétase (DAPT) diminue la production d’Aβ1-42 intracellulaire et restaure la survie neuronale. Ces résultats indiquent que l’accumulation intraneuronale d’Aβ1-42 est responsable de la neurotoxicité observée dans notre modèle.
Afin de mieux caractériser cette neurotoxicité, nous avons mesuré l’activité d’un effecteur de l’apoptose, la caspase 3. L’expression de l’APP humain induit de manière importante l’activité de la caspase 3. Les neurones traités par la DAPT présentent une activité de la caspase 3 réduite par rapport à celle des neurones non-traités. Ceci suggère que l’accumulation d’Aβ1-42 intracellulaire active la caspase 3.
Cependant, le traitement des neurones exprimant l’APP humain avec un inhibiteur spécifique de la caspase 3 (Z-DEVD-FMK) n’exerce aucun effet sur la survie neuronale. La caspase 3 ne semble donc pas être un effecteur majeur de la neurotoxicité observée dans notre modèle

Neurotoxicity Syndromes --- Amyloid beta-Peptides --- Blotting, Western --- Enzyme-Linked Immunosorbent Assay

Choose an application

• Reference Manager
• EndNote
• RefWorks (Direct export to RefWorks)

Protein blotting techniques have become common laboratory procedures in the past few years. This text is written by scientists with expertise in these techniques. The versatility of the methods utilizing these procedures have brought about the development of different solid support matrices, as well as a wide variety of protein detection methods. While the most commonly used method in protein blotting is the use of antibodies to detect protein antigens, this technology has been expanded to examine a number of different interactions between proteins and other proteins, as well as other molecules such as carbohydrates and DNA. These methods have further been adapted for amino acid sequencing and purification of proteins for use as immunogens. This book outlines, in detail, numerous protocols and procedures which should help investigators design methods which will be optimal for their specific use.

Western immunoblotting --- Blotting, Western. --- Proteins --- 577.112 --- 57.088.1 --- 57.088.1 Methods for analysis and estimation --- Methods for analysis and estimation --- 577.112 Proteins --- Blot, Western --- Immunoblot, Western --- Western Blot --- Western Immunoblot --- Immunoblotting, Western --- Western Blotting --- Western Immunoblotting --- Blots, Western --- Blottings, Western --- Immunoblots, Western --- Immunoblottings, Western --- Western Blots --- Western Blottings --- Western Immunoblots --- Western Immunoblottings --- Protein blotting --- Western blot analysis --- Western blotting --- Immunoblotting --- Laboratory manuals. --- analysis. --- Analysis --- Blotting, Western --- Laboratory manuals --- analysis --- Analysis. --- Proteins - analysis --- Western immunoblotting - Laboratory manuals

Choose an application

• Reference Manager
• EndNote
• RefWorks (Direct export to RefWorks)

Several methods (chromatographic analysis, Western ligand-blotting, radioimmunoassay) have been developed, aiming in a first time to characterize bovine IGFBP's, plasma proteins that specifically bind insulin-like growth factors (IGF's) and, in a second time, to quantitatively determine some of them. These methods were validated by the study in the male calve of the evolution of IGFBP's during the peripubertal period, by the observation in the male and female cattle of the effect of modification of nutritional status on IGFBP's, and finally by the study of the implication of IGFBP-2 and -3 in the influence exerted by severe or moderate feed-restriction on the stablishment of puberty in the bull calf. The obtained results show the efficacy of the radioimmunoassay developped to determine the IGFBP-2 plasma concentration, which opens different perspectives for further works applied to the agronomical field (determination of the age at puberty, investigation of growth hormone treatment, etc.).

Cattle --- Cattle --- Young animals --- Young animals --- Insulin --- Insulin --- Binding proteins --- Binding proteins --- animal growth promoters --- animal growth promoters --- Immunoenzyme techniques --- Immunoenzyme techniques --- Chromatography --- Chromatography --- Radiobiology --- Radiobiology --- Western ligand-blotting --- Western ligand-blotting