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

Liang, Yixiu; Yao, Yuwei; Lu, Ming‐Xing; Hou, Jiapeng; Yu, Shenliang; Lu, Boxun

doi:10.3233/jhd-140104

Cited by 9 publications

(17 citation statements)

References 6 publications

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“…Gpr52 siRNAs were tranfected for 3 days, whereas the compound treatment (forskolin: 1 µM; reserpine: 10 µM) lasts for 24 hr; statistical analyses performed by the two-tailed Mann–Whitney U test. ( B ) Htt level measured by the 2B7/2166 HTRF ( Liang et al, 2014 ) upon treatment of different doses of the Gpr52 agonist reserpine for 48 hr, with transfection of Gpr52 siRNA (Gpr52_si1) vs the non-targeting control (Neg_si), n = 4. ( C ) Htt level measured by the 2B7/2166 HTRF when transfected with hGPR52 cDNA titrated with the empty control vector at different percentages (X-axis), n = 6; statistical analysis performed by the one-way ANOVA and post-hoc Dunnett's test.…”

Section: Resultsmentioning

confidence: 99%

“…Calnexin has been used as a loading control. The Bars plot : Htt level changes (%) measured by 2B7/2166 HTRF ( Liang et al, 2014 ) of the total lysates with the same treatments as in the western-blot samples. ( E ) Confocal microscopy experiments showing that HTT proteins are enriched in the perinuclear and co-localize with the endoplasmic reticulum (ER) marker calreticulin upon treatment of Rp-cAMP or 8-pCpt-2′-O-Me-cAMP (8-pCpt-cAMP for short).…”

Section: Resultsmentioning

confidence: 99%

“…The HTRF assays were performed similarly to those previously described ( Lu et al, 2013 ; Liang et al, 2014 ). For all the samples, the protein concentration (by BCA, Life Technologies, cat.…”

Section: Methodsmentioning

confidence: 99%

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A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity

Yao

Cui

Al‐Ramahi

et al. 2015

eLife

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Huntington's disease (HD) represents an important model for neurodegenerative disorders and proteinopathies. It is mainly caused by cytotoxicity of the mutant huntingtin protein (Htt) with an expanded polyQ stretch. While Htt is ubiquitously expressed, HD is characterized by selective neurodegeneration of the striatum. Here we report a striatal-enriched orphan G protein-coupled receptor(GPCR) Gpr52 as a stabilizer of Htt in vitro and in vivo. Gpr52 modulates Htt via cAMP-dependent but PKA independent mechanisms. Gpr52 is located within an intron of Rabgap1l, which exhibits epistatic effects on Gpr52-mediated modulation of Htt levels by inhibiting its substrate Rab39B, which co-localizes with Htt and translocates Htt to the endoplasmic reticulum. Finally, reducing Gpr52 suppresses HD phenotypes in both patient iPS-derived neurons and in vivo Drosophila HD models. Thus, our discovery reveals modulation of Htt levels by a striatal-enriched GPCR via its GPCR function, providing insights into the selective neurodegeneration and potential treatment strategies.DOI: http://dx.doi.org/10.7554/eLife.05449.001

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity

Yao

Cui

Al‐Ramahi

et al. 2015

eLife

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“…HTRF detects HTT proteins by measuring the time-resolved fluorescent resonance energy transfer (TR-FRET) between two HTT antibodies labeled with either fluorescent donor or acceptor molecules 9 . We have used the 2B7/MW1 antibody pair to detect the mHTT in the HD patient fibroblast cells 11 , the 2B7/2166 antibody pair to detect the wild-type HTT (wtHTT) in the control patient fibroblasts 12 , or both the wtHTT and mHTT in the mouse striatal cells STHdh Q7/Q111 9 . The effort of replacing the Western blot directly with the high-throughput-compatible HTRF assay failed because of two major reasons ( Figure 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…HTRF assays were performed similarly to those previously described 2 , 3 , 9 . The antibody mixtures were prepared by adding the donor and acceptor antibodies into the HTRF assay buffer containing 50 mmol/L NaH 2 PO 4 , 400 mmol/L KF, 0.1% bovine serum albumin (BSA), and 0.05% Tween 20.…”

Section: Methodsmentioning

confidence: 99%

A high-throughput-compatible assay to measure the degradation of endogenous Huntingtin proteins

Cui

et al. 2016

Acta Pharmacol Sin

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Aim:The accumulation of disease-causing proteins is a common hallmark of many neurodegenerative disorders. Measuring the degradation of such proteins using high-throughput-compatible assays is highly desired for the identification of genetic and chemical modulators of degradation. For example, Huntington's disease (HD) is an incurable hereditary neurodegenerative disorder caused by the cytotoxicity of mutant huntingtin protein (mHTT). The high-throughput measurement of mHTT degradation is important in HD drug discovery and research. Existing methods for such purposes have limitations due to their dependence on protein tags or pan protein synthesis inhibitors. Here, we report a high-throughput-compatible pulse-chase method (CH-chase) for the measurement of endogenous tag-free huntingtin protein (HTT) degradation based on Click chemistry and Homogeneous Time Resolved Fluorescence (HTRF) technologies.Methods:The pulsed-labeled proteins were conjugated with biotin using the click reaction strain-promoted alkyne-azide cycloaddition (SPAAC), and the chase signals were calculated by measuring the reduction percentage of the HTT HTRF signals after pull-down with streptavidin beads.Results:We validated that the signals were within the linear detection range and were HTT-specific. We successfully measured the degradation of endogenous HTT in a high-throughput-compatible format using 96-well plates. The predicted changes of HTT degradation by known modifiers were observed, which confirmed that the assay is suitable for the identification of HTT degradation modifiers.Conclusion:We have established the first high-throughput-compatible assay capable of measuring endogenous, tag-free HTT degradation, providing a valuable tool for HD research and drug discovery. The method could be applied to other proteins and can facilitate research on other neurodegenerative disorders and proteinopathies.

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Exploiting the Unique Properties of Lanthanide Complexes as FRET Probes: from Quantitation to Protein Dynamics

Wheeler

Butler

2022

Analysis & Sensing

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Lanthanide complexes have a range of photophysical properties -large pseudo-Stokes shifts, narrow linewidths, long emission lifetimes -that make them ideal as emissive probes. These same properties also render them excellent donors in assays relying on Förster resonance energy transfer (FRET). Over the last 20 years these assays have increased in sophistication so that a range of biological applications has now been demonstrated. These range from easy-to-use quantitation of proteins (a practical alternative to western blotting) to sophisticated experiments which give real-time information on the movement of proteins or allow simultaneous readout of both agonist and co-activator binding to a protein. We introduce the principles and difficulties of using such assays before surveying the full scope of what they can achieve and attempting to anticipate future developments in this area.

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

Cited by 9 publications

References 6 publications

A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity

A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity

A high-throughput-compatible assay to measure the degradation of endogenous Huntingtin proteins

Exploiting the Unique Properties of Lanthanide Complexes as FRET Probes: from Quantitation to Protein Dynamics

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