A High-Throughput Fluorescence Polarization-Based Assay for the SH2 Domain of 
STAT4

Berg, Angela; Gräber, Martin; Schmutzler, Sebastian; Hoffmann, Ralf; Berg, Thorsten

doi:10.3390/mps5060093

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A High-Throughput Fluorescence Polarization-Based Assay for the SH2 Domain of STAT4

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Gräber, Martin
Ullrich, Axel / Molecular Biology, Max Planck Institute of Biochemistry, Max Planck Society;

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Citation

Berg, A., Gräber, M., Schmutzler, S., Hoffmann, R., & Berg, T. (2022). A High-Throughput Fluorescence Polarization-Based Assay for the SH2 Domain of STAT4. Methods and protocols, 5(6): 93. doi:10.3390/mps5060093.

Cite as: https://hdl.handle.net/21.11116/0000-000C-754B-D

Abstract

The signal transducer and activation of transcription (STAT) proteins are a family of Src homology 2 (SH2) domain-containing transcription factors. The family member STAT4 is a mediator of IL-12 signalling and has been implicated in the pathogenesis of multiple autoimmune diseases. The activity of STAT4 requires binding of phosphotyrosine-containing motifs to its SH2 domain. Selective inhibitors of the STAT4 SH2 domain have not been published to date. Here, we present a fluorescence polarization-based assay for the identification of inhibitors of the STAT4 SH2 domain. The assay is based on the interaction between the STAT4 SH2 domain and the fluorophore-labelled peptide 5-carboxyfluorescein-GpYLPQNID (K-d = 34 +/- 4 nM). The assay is stable with respect to DMSO concentrations of up to 10% and incubation times of at least 8 h. The Z'-value of 0.85 +/- 0.01 indicates that the assay is suited for use in high-throughput screening campaigns aimed at identifying new therapeutic modalities for the treatment of autoimmune diseases.