Name of Authors: Geneviève Chatel, Mireille Caron and Jaime Padros
Name of Institution: BioAuxilium
The Signal Transducer and Activator of Transcription (STAT) family of proteins plays a complex and
essential role in mediating cellular responses to cytokines and growth factors. The abnormal activation
of STAT proteins, including STAT1, STAT3 and STAT6, has been associated with malignant transformation.
Therefore, the ability to assess the phosphorylation of STAT1, STAT3 and STAT6 in a cellular setting is
important for drug discovery research. The homogeneous immunoassay formats available to detect and
quantify these STAT proteins in cell lysates are expensive, thereby precluding its wide adoption in
academic and small industrial laboratories. Here we describe the validation of improved immunoassays
for the measurement in a 384-well format of phosphorylated STAT1 (Y701), STAT3 (Y705) and STAT6
(Y641) in cell lysates using the THUNDER TR-FRET platform. These optimized cell-based assays were
applied for determining the pharmacological profile of known activators and inhibitors of the JAK/STAT
pathway. All assays exhibited wide dynamic ranges, high signal-to-background-ratios and Z’ values higher
than 0.8. Overall, the results presented here demonstrate that the novel THUNDER phospho-STAT1,
phospho-STAT3 and phospho-STAT6 TR-FRET assays generate robust, reproducible data and are
amenable to high-throughput screening (HTS) applications. This suite of homogeneous assays provides
new tools for the screening and characterization of specific and selective modulators of the JAK/STAT
signaling pathway. The THUNDER TR-FRET platform represents a cost-effective alternative to traditional
homogeneous immunoassays, both in an academic setting and in HTS laboratories.
e-mail: jaime.padros@bioauxilium.com