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Updated: Jul 10, 2026

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Published on: March 3, 2015

High-throughput cellular assays for regulated posttranslational modifications.

Matthew B Robers¹, Robert A Horton, Mark R Bercher

¹Invitrogen Discovery Sciences, Madison, WI 53717, USA.

Analytical Biochemistry

|October 27, 2007

Summary

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We developed high-throughput screening assays to measure posttranslational modifications in mammalian cells. These assays track NF-kappaB pathway signaling, aiding drug discovery by analyzing cellular processes.

Area of Science:

Cellular biology
Molecular signaling
Assay development

Background:

Posttranslational modifications regulate numerous cellular processes.
The NF-kappaB signaling pathway is crucial in immunity and disease and involves complex posttranslational modifications.
Existing methods for studying these modifications are often low-throughput and lack physiological relevance.

Purpose of the Study:

To develop and validate high-throughput screening (HTS)-compatible assays for measuring target-specific posttranslational modifications in mammalian cells.
To utilize the NF-kappaB signaling cascade as a model system to demonstrate the utility of these novel assays.
To provide tools for dissecting intermediate steps in signaling pathways and facilitating target-based drug discovery.

Main Methods:

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High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines

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Last Updated: Jul 10, 2026

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Published on: March 3, 2015

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Published on: November 7, 2019

High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines

High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines

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Developed stable cell lines expressing IkappaBalpha fused to green fluorescent protein (GFP).
Utilized time-resolved Förster resonance energy transfer (TR-FRET) with terbium-labeled antibodies to measure IkappaBalpha phosphorylation and ubiquitination.
Engineered a beta-lactamase-IkappaBalpha reporter cell line to quantify proteasomal degradation of IkappaBalpha in living cells.

Main Results:

Successfully established HTS-compatible assays for monitoring key posttranslational modifications in the NF-kappaB pathway.
Demonstrated the ability to measure phosphorylation, ubiquitination, and proteasomal degradation of IkappaBalpha in a chronological manner.
Validated the sensitivity and robustness of both TR-FRET and beta-lactamase reporter assays in a cellular context.

Conclusions:

The developed TR-FRET and beta-lactamase reporter assays offer a convenient, sensitive, and robust method for studying NF-kappaB signaling.
These assays provide a physiologically relevant cellular context without requiring overexpression of pathway enzymes.
The HTS assays are broadly applicable for pathway analysis and target-based drug discovery.