Time-resolved Förster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

Summary

Time-resolved Förster resonance energy transfer cell-based assay protocols are described for the simple, specific, sensitive, and robust quantification of endogenous phosphorylated signal transducer and activator of transcription (STAT) 1/3/4/5/6 proteins in cell lysates in a 384-well format.

Abstract

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to cytokines and growth factors. STAT proteins are activated by tyrosine phosphorylation mediated mainly by JAKs. The abnormal activation of STAT signaling pathways is implicated in many human diseases, especially cancer and immune-related conditions. Therefore, the ability to monitor STAT protein phosphorylation within the native cell signaling environment is important for both academic and drug discovery research. The traditional assay formats available to quantify phosphorylated STAT proteins include western blotting and the enzyme-linked immunosorbent assay (ELISA). These heterogeneous methods are labor-intensive, low-throughput, and often not reliable (specific) in the case of western blotting. Homogeneous (no-wash) methods are available but remain expensive.

Here, detailed protocols are provided for the sensitive, robust, and cost-effective measurement in a 384-well format of endogenous levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641) in cell lysates from adherent or suspension cells using the novel THUNDER time-resolved Förster resonance energy transfer (TR-FRET) platform. The workflow for the cellular assay is simple, fast, and designed for high-throughput screening (HTS). The assay protocol is flexible, uses a low-volume sample (15 µL), requires only one reagent addition step, and can be adapted to low-throughput and high-throughput applications. Each phospho-STAT sandwich immunoassay is validated under optimized conditions with known agonists and inhibitors and generates the expected pharmacology and Z'-factor values. As TR-FRET assays are ratiometric and require no washing steps, they provide much better reproducibility than traditional approaches. Together, this suite of assays provides new cost-effective tools for a more comprehensive analysis of specific phosphorylated STAT proteins following cell treatment and the screening and characterization of specific and selective modulators of the JAK/STAT signaling pathway.

Introduction

The JAK/STAT signaling pathway plays a key role in mediating cellular responses to diverse cytokines, interferons, growth factors, and related molecules^1,2. The binding of these ligands to specific cell-surface receptors results in the activation of JAKs, which in turn activate STAT proteins by phosphorylation of specific tyrosine residues. STAT phosphorylation results in their dimerization and translocation into the nucleus, where they exert their effect on the transcription of regulated target genes. The STAT family consists of seven members: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. The members....

Protocol

1. Cell culture

1. Maintain cells in a humidified 37 °C/5% CO₂ incubator and culture with either DMEM supplemented with 10% fetal bovine serum (FBS) (HeLa and A431 cells) or RPMI supplemented with 15% FBS (U266B1 cells). Culture the cells until they reach 70-80% confluence, then trypsinize them and passage or use them for the assays.
   ​NOTE: Culture media contained phenol red. No serum starvation was conducted for any cell line prior to conducting the assays.

2. Stimulator or inhibitor titration using the two-plate assay protocol with adherent cells

NOTE: This procedure describes how ....

Results

Each THUNDER TR-FRET assay was pharmacologically validated by treating adherent (HeLa or A431) or suspension cells (U266B1) with JAK/STAT pathway-specific activators or inhibitors and then measuring the levels of specific phosphorylated and total STATs, when applicable. Assays were conducted in 384-well format using the two-plate transfer protocol and pre-optimized assay conditions. Figure 3, Figure 4, Figure 5,

Discussion

Compared to conventional methods for phosphoprotein analysis such as western blotting and ELISA-based methods, the workflow for a THUNDER TR-FRET cellular assay is simple and fast, uses a low-volume sample (15 µL), is designed for HTS in a 384-well format, and is highly amenable to automation. The assay protocol is flexible and can readily be adapted to both medium- and high-throughput applications. Assays can be run using either a two-plate transfer protocol or a one-384-well plate protocol. In the two-plate transf.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well microplate, clear, flat bottom, polystyrene, tissue culture-treated, sterile	Corning	3595	This is the plate for culturing cells when using the two-plate assay protocol. Other cell culture 96-well plates can be used
384-well microplate, white, low-volume	PerkinElmer	6007290	This is the plate for TR-FRET detection when using the two- or one-plate assay protocols. Other low-volume, white 384-well plates can be used
A431 cell line	ATCC	CRL-1555
Adhesive microplate seal	PerkinElmer	6050185
DMSO	Fisher	D159-4
Dulbecco’s modified Eagle medium (DMEM)	Wisent	319-005-CL	THUNDER TR-FRET is compatible with culture medium containing phenol red
EnVision Xcite Multilabel plate reader	PerkinElmer	2104-0020A	The assays can be performed on a variety of plate readers equipped with the TR-FRET option
Erlotinib hydrochloride	Sigma	CDS022564
Falcon tissue culture treated flasks	Fisher	13-680-65
Fetal bovine serum (FBS)	Wisent	098-150
HeLa cell line	ATCC	CCL-2
JAK Inhibitor 1	Cayman Chemical	15146
Orbital plate shaker	Many options available	Not applicable
Recombinant human EGF	PeproTech	AF-100-15
Recombinant human IFNα2b	ProSpec	CYT-460
Recombinant human IL-4	R&D Systems	204-IL
Roswell Park Memorial Institute 1640 medium (RPMI)	Wisent	350-007-CL	THUNDER TR-FRET is compatible with culture medium containing phenol red
THUNDER Phospho-STAT1 (Y701) + Total STAT1 TR-FRET Cell Signaling Assay Kit	BioAuxilium Research	KIT-STAT1PT-500
THUNDER Phospho-STAT3 (Y705) + Total STAT3 Cell Signaling Assay Kit	BioAuxilium Research	KIT-STAT3PT-500
THUNDER Phospho-STAT4 (Y693) TR-FRET Cell Signaling Assay Kit	BioAuxilium Research	KIT-STAT4P-500
THUNDER Phospho-STAT5 (Y694/Y699) + Total STAT5 TR-FRET Cell Signaling Assay Kit	BioAuxilium Research	KIT-STAT5PT-500
THUNDER Phospho-STAT6 (Y641) + Total STAT6 TR-FRET Cell Signaling Assay Kit	BioAuxilium Research	KIT-STAT6PT-500
Trypsin/EDTA 0.05%	Wisent	325-542-CL
U266B1 cell line	ATCC	TIB-196
Ultrapure water	NA	NA	Use Milli-Q grade water (18 MΩ.cm) to dilute Lysis Buffer and Detection Buffer