TR-FRET Assays for Endogenous Huntingtin Protein Level in Mouse Cells.

J Huntingtons Dis

State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China Mass General Institute for Neurodegenerative Disease, Charlestown, MA, USA.

Published: February 2016

High-throughput measurement of huntingtin (Htt) levels is useful for Huntington's disease research. For example, identification of genetic or chemical modifiers that reduce Htt levels by high-throughput screening provides promising strategy for HD drug discovery. In the human cells, high-throughput measurement of Htt levels has been established based on the Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) technology, using the 2B7/MW1 antibody pair. Unfortunately, application of this assay in the mouse cells has been problematic due to discrepancies between TR-FRET signals and Western-blots, possibly caused by non-specific antibody binding. Here we report TR-FRET assays that are able to detect endogenous Htt levels of the mouse striatal cell line (STHdh).

Download full-text PDF

Source
http://dx.doi.org/10.3233/JHD-140104DOI Listing

Publication Analysis

Top Keywords

htt levels
16
tr-fret assays
8
mouse cells
8
cells high-throughput
8
high-throughput measurement
8
tr-fret
4
assays endogenous
4
endogenous huntingtin
4
huntingtin protein
4
protein level
4

Similar Publications

Polycystic ovarian syndrome (PCOS) is a complex endocrine-metabolic disorder, and multiple factors contribute to its pathophysiology. The current study assessed a PCOS-like animal model induced by consuming a high-fat sugar (HFHS) diet and compared the treatment outcome of mitochondrial-targeted antioxidants versus heat therapy. Sixty rats were divided into the following study groups: three control groups (negative and positive for the treatments used), HFHS, hot tub therapy (HTT) treatment, and MitoQ10 treatment (500 µmol/L MitoQ10 in clean drinking water daily, from week fourteen till week twenty-two of the study).

View Article and Find Full Text PDF

Objective: Psychosocial and genetic factors are considered to play roles in the etiological mechanisms of major depressive disorder (MDD). The involvement of miRNAs in the etiopathogenesis of depression and childhood traumas is still unclear. This study aims to reveal potential differences in miRNA levels between patients with depression and healthy individuals and assess their connection to childhood traumas.

View Article and Find Full Text PDF

RNA-Targeting CRISPR/CasRx system relieves disease symptoms in Huntington's disease models.

Mol Neurodegener

January 2025

Guangdong Key Laboratory of Non-Human Primate Research, Key Laboratory of CNS Regeneration (Ministry of Education), School of Medicine, GHM Institute of CNS Regeneration, Jinan University, Guangzhou, 510632, China.

Background: HD is a devastating neurodegenerative disorder caused by the expansion of CAG repeats in the HTT. Silencing the expression of mutated proteins is a therapeutic direction to rescue HD patients, and recent advances in gene editing technology such as CRISPR/CasRx have opened up new avenues for therapeutic intervention.

Methods: The CRISPR/CasRx system was employed to target human HTT exon 1, resulting in an efficient knockdown of HTT mRNA.

View Article and Find Full Text PDF

has been identified in human and mouse HD brain as the pathogenic exon 1 mRNA generated from aberrant splicing between exon 1 and 2 that contributes to aggregate formation and neuronal dysfunction (Sathasivam et al., 2013). Detection of the HTT exon 1 protein (HTTex1p) has been accomplished with surrogate antibodies in fluorescence-based reporter assays (MSD, HTRF), and immunoprecipitation assays, in HD postmortem cerebellum and knock-in mice but direct detection by SDS-PAGE and western blot assay has been lacking.

View Article and Find Full Text PDF

To elucidate the potential roles of presynaptic and postsynaptic serotonergic activity in impulsivity traits, we investigated the relationship between self-reported impulsiveness and serotonin transporter (5-HTT) and 5-HT2A receptors in healthy individuals. In this study, 26 participants completed 3-Tesla magnetic resonance imaging and positron emission tomography with [C]DASB and [C]MDL100907. To quantify 5-HTT and 5-HT2A receptor availability, the binding potential (BP) of [C]DASB and [C]MDL100907 was derived using the simplified reference tissue model with cerebellar gray matter as the reference region.

View Article and Find Full Text PDF

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!