1. Cell culture
<ol>
	<li>Maintain cells in a humidified 37 &#176;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. 
	&#8203;NOTE: Culture media contained phenol red. No serum starvation was conducted for any cell line prior to conducting the assays.</li>
</ol>
<p class="jove...

<ol>
	<li>Villarino, A., Kanno, Y., O'Shea, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+consequences+of+Jak-STAT+signaling+in+the+immune+system.">Mechanisms and consequences of Jak-STAT signaling in the immune system.</a> Nature Immunology. 18 (4), 374-384 (2017).</li><li>Hammar&#233;n, H. M., Virtanen, A. T., Raivola, J., Silvennoinen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+JAKs+in+cytokine+signaling+and+its+breakdown+in+disease.">The regulation of JAKs in cytokine signaling and its breakdown in disease.</a> Cytokine. 118, 48-63 (2019).</li><li>O'Shea, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+JAK-STAT+pathway:+impact+on+human+disease+and+therapeutic+intervention.">The JAK-STAT pathway: impact on human disease and therapeutic intervention.</a> Annual Review of Medicine. 66, 311-328 (2015).</li><li>Verhoeven, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al.+potential+and+controversy+of+targeting+STAT+family+members+in+cancer.">et al. potential and controversy of targeting STAT family members in cancer.</a> Seminars in Cancer Biology. 60 (2), 41-56 (2020).</li><li>Gilda, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al.+blotting+inaccuracies+with+unverified+antibodies:+need+for+a+Western+blotting+minimal+reporting+standard+(WBMRS).">et al. blotting inaccuracies with unverified antibodies: need for a Western blotting minimal reporting standard (WBMRS).</a> PLoS One. 10 (8), 0135392 (2015).</li><li>Binder, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al.+and+utilization+of+the+SureFire+phospho-STAT5+assay+for+a+cell-based+screening+campaign.">et al. and utilization of the SureFire phospho-STAT5 assay for a cell-based screening campaign.</a> Assay and Drug Development Technologies. 6 (1), 27-37 (2008).</li><li>Ayoub, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogeneous+time-resolved+fluorescence-based+assay+to+monitor+extracellular+signal-regulated+kinase+signaling+in+a+high-throughput+format.">Homogeneous time-resolved fluorescence-based assay to monitor extracellular signal-regulated kinase signaling in a high-throughput format.</a> Frontiers in Endocrinology. 5, 94 (2014).</li><li>Robers, M. B., Machleidt, T., Carlson, C. B., Bi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+LanthaScreen+and+beta-lactamase+reporter+assays+for+high-throughput+screening+of+JAK2+inhibitors.">Cellular LanthaScreen and beta-lactamase reporter assays for high-throughput screening of JAK2 inhibitors.</a> Assay and Drug Development Technologies. 6 (4), 519-529 (2008).</li><li>Hwang, B., Engel, L., Goueli, S. A., Zegzouti, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogeneous+bioluminescent+immunoassay+to+probe+cellular+signaling+pathway+regulation.">Homogeneous bioluminescent immunoassay to probe cellular signaling pathway regulation.</a> Communications Biology. 3 (8), 1-12 (2020).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+statistical+parameter+for+use+in+evaluation+and+validation+of+high-throughput+screening+assays.">A simple statistical parameter for use in evaluation and validation of high-throughput screening assays.</a> Journal of Biomolecular Screening. 4 (2), 67-73 (1999).</li><li>Osmond, R. I. W., Das, S., Crouch, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+cell-based+assays+for+cytokine+receptor+signaling,+using+an+AlphaScreen+SureFire+assay+format.">Development of cell-based assays for cytokine receptor signaling, using an AlphaScreen SureFire assay format.</a> Analytical Biochemistry. 403 (1-2), 94-101 (2010).</li><li>Haan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jak1+has+a+dominant+role+over+Jak3+in+signal+transduction+through+&#947;c-containing+cytokine+receptors.">Jak1 has a dominant role over Jak3 in signal transduction through &#947;c-containing cytokine receptors.</a> Chemistry &amp; Biology. 18 (3), 314-323 (2011).</li><li>Kim, Y., Apetri, M., Luo, B., Settleman, J. E., Anderson, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+tyrosine+kinase+inhibitors+on+normal+and+oncogenic+EGFR+signaling+and+downstream+effectors.">Differential effects of tyrosine kinase inhibitors on normal and oncogenic EGFR signaling and downstream effectors.</a> Molecular Cancer Research. 13 (4), 765-774 (2015).</li><li>Qian, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+homogeneous+cell-based+kinase+assays+for+JAK2+V617F:+SureFire+pSTAT5+and+GeneBLAzer+fluorescence+resonance+energy+transfer+assays.">Comparison of two homogeneous cell-based kinase assays for JAK2 V617F: SureFire pSTAT5 and GeneBLAzer fluorescence resonance energy transfer assays.</a> ASSAY and Drug Development Technologies. 10 (2), 212-217 (2012).</li><li>Iversen, P. W., et al., Markossian, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HTS+assay+validation.">HTS assay validation.</a> Assay Guidance Manual. , (2012).</li><li>Muelas, M. W., Ortega, F., Breitling, R., Bendtsen, C., Westerhoff, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+cell+culture+optimization+enhances+experimental+reproducibility+in+cancer+cells.">Rational cell culture optimization enhances experimental reproducibility in cancer cells.</a> Scientific Reports. 8, 3029 (2018).</li></ol>

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 &#181;L), 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...

The JAK/STAT signaling pathway plays a key role in mediating cellular responses to diverse cytokines, interferons, growth factors, and related molecules1,2. 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 play a complex and essential role in the regulation of physiologic cell processes, including proliferation, differentiation, apoptosis, angiogenesis, and immune system regulation. The abnormal activation of STAT signaling pathways is implicated in many human diseases, especially cancer and immune-related conditions3,4. Therefore, the ability to assess STAT protein phosphorylation within the native cell signaling environment is important for both academic and drug discovery research.
To date, the conventional methods used to measure intracellular phosphorylated protein levels, including STATs, are antibody-based and include western blotting, ELISA, and phosphoflow cytometry. These heterogeneous methods are labor-intensive, time-consuming, error-prone, low-throughput, and often unreliable (e.g., specificity issues) in the case of western blotting5. In contrast, homogeneous assays require fewer experimental steps, use smaller sample volumes, and are amenable to HTS. There are five homogeneous cell-based immunoassay platforms commercially available that can be used to quantitatively monitor JAK-dependent phosphorylation of STATs in cell lysates: SureFire, HTRF, LANCE, LanthaScreen, and Lumit. Each of these platforms has its advantages and disadvantages.
SureFire is based on luminescent oxygen channeling technology, which utilizes donor and acceptor beads coated to specifically capture a pair of antibodies, one of which is biotinylated. In the presence of phosphorylated protein, the two antibodies bring the donor and acceptor beads into close proximity, enabling the generation of a chemiluminescent signal6. While versatile and sensitive, this technology is expensive, is affected by biotin in the culture medium, is very sensitive to ambient temperature and light, and requires a special reader for detection. HTRF and LANCE are both based on TR-FRET technology that utilizes long-lifetime luminescent lanthanide ion complexes (Europium or Terbium chelates, or Europium cryptate) as the donor molecules and far-red fluorophores as the acceptor molecules7. When two protein-specific antibodies labeled with either donor or acceptor molecules are brought into close proximity, FRET takes place, causing an increase in acceptor fluorescence and a decrease in donor fluorescence. These long-lived fluorescent signals can be measured in a time-resolved and ratiometric manner to reduce assay interference and increase data quality. Other advantages of TR-FRET are that it is not light-sensitive, allows repeated readings, and exhibits long signal stability. While TR-FRET is widely implemented in HTS due to its versatility, sensitivity, and high robustness, all commercial TR-FRET-based assay platforms are expensive, thereby precluding its wide adoption in academic and small industrial laboratories. The LanthaScreen assay also uses a TR-FRET based-readout but is reliant on an engineered U2OS cell line that stably expresses green fluorescent protein (GFP)-STAT1 fusion protein combined with a terbium-labeled phospho-specific STAT1 antibody8. In addition to being limited in terms of choice of signaling proteins, this method requires purchasing expensive transfected cell lines, reducing its applicability and increasing the possibility of experimental artifacts. Lumit is a generic bioluminescent immunoassay platform that utilizes secondary antibodies (anti-mouse and anti-rabbit) chemically labeled with the small and large NanoBit subunits of NanoLuc Luciferase9. The binding of two primary antibodies to the target protein brings the secondary antibodies into proximity to form an active enzyme that generates a luminescence signal. While luminescence is generally a sensitive and robust readout, the requirement for primary antibodies raised in two different species limits the choices for assay design. In addition, the use of secondary antibodies in complex sample matrixes may be prone to assay interference.
Thus, a need still exists for a reliable, rapid, yet affordable cell-based assay platform for measuring individual phosphorylated and total STAT proteins in a manner compatible with HTS. To address this need, a new high-throughput cell-based immunoassay platform was developed based on an enhanced TR-FRET technology (THUNDER) and designed to enable simple, sensitive, robust, and cost-effective measurement of endogenously expressed intracellular proteins (phosphorylated or total) in cell lysates. The advantages of this technology stem from the combination of a donor/acceptor FRET pair exhibiting exceptional spectral compatibility and TR-FRET signal, rigorously validated antibodies, and optimized lysis buffers. These assays are formatted as sandwich immunoassays and use a straightforward, three-step workflow (Figure 1). Cells are first treated to modulate protein phosphorylation and then lysed with the specific lysis buffer provided in the kit. The target phosphorylated or total STAT protein in the cell lysate is detected in a single reagent addition and incubation step with a pair of fluorophore-labeled antibodies that recognize distinct epitopes on the target protein (Figure 2). One antibody is labeled with a Europium chelate donor (Eu-Ab1), while the second antibody is labeled with a far-red acceptor fluorophore (FR-Ab2). The two labeled antibodies bind to the protein in solution, bringing the two labels into close proximity. Excitation of the donor Europium chelate at 320 or 340 nm triggers a FRET to the acceptor, which emits a long-lived TR-FRET signal at 665 nm proportional to the concentration of target protein (phosphorylated or total) in the cell lysate.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62915/62915fig01.jpg" /> 
Figure 1: TR-FRET assay workflow. The workflow consists of three steps: cell treatment, cell lysis, and protein detection using TR-FRET. In the two-plate assay protocol, lysates are transferred to a white 384-well detection plate, whereas in the one-plate protocol, all steps are conducted in the same white 384-well detection plate (all-in-one-well protocol). Regardless of the assay protocol used, protein detection is performed in the same total volume (20 &#181;L per well). Abbreviation: TR-FRET = time-resolved F&#246;rster resonance energy transfer. <a href="https://www.jove.com/files/ftp_upload/62915/62915fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62915/62915fig02.jpg" /> 
Figure 2: TR-FRET sandwich immunoassay principle. One antibody is labeled with the Europium chelate donor (Eu-Ab1) and the second with the far-red small fluorophore acceptor (FR-Ab2). The two labeled antibodies bind specifically to distinct epitopes on the target protein (phosphorylated or total) in the cell lysate, bringing the two fluorophores into close proximity. Excitation of the donor Europium chelate at 320 or 340 nm triggers a FRET from the donor to the acceptor molecules, which in turn emit a signal at 665 nm. This signal is proportional to the concentration of protein in the cell lysate. In the absence of the specific target protein, the donor and acceptor fluorophores are too distant from each other for FRET to occur. Abbreviations: FRET = F&#246;rster resonance energy transfer; TR-FRET = time-resolved FRET; Ab = antibody; FR = far-red; Eu - Europium chelate; P = phosphorylation. <a href="https://www.jove.com/files/ftp_upload/62915/62915fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Here, detailed protocols are provided for measuring, in a 384-well format, the intracellular levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641), together with total STAT1, STAT3, STAT5, and STAT6, in cell lysates from adherent or suspension cells using the THUNDER TR-FRET platform. These protocols define steps for cell treatment, lysis, and TR-FRET-based target protein detection using either a two-plate transfer protocol or a one-plate all-in-one-well protocol. These cell-based assays are applied for determining the pharmacological profile of known activators and inhibitors of the JAK/STAT pathway. The robustness and suitability of selected assays for HTS are demonstrated. Lastly, key experiments for assay optimization are discussed, along with recommendations for assay troubleshooting.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well microplate, clear, flat bottom, polystyrene, tissue culture-treated, sterile</td>
			<td>Corning</td>
			<td>3595</td>
			<td>This is the plate for culturing cells when using the two-plate assay protocol. Other cell culture 96-well plates can be used</td>
		</tr>
		<tr>
			<td>384-well microplate, white, low-volume</td>
			<td>PerkinElmer</td>
			<td>6007290</td>
			<td>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</td>
		</tr>
		<tr>
			<td>A431 cell line</td>
			<td>ATCC</td>
			<td>CRL-1555</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive microplate seal</td>
			<td>PerkinElmer</td>
			<td>6050185</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher</td>
			<td>D159-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle medium (DMEM)</td>
			<td>Wisent</td>
			<td>319-005-CL&#160;</td>
			<td>THUNDER TR-FRET is compatible with culture medium containing phenol red</td>
		</tr>
		<tr>
			<td>EnVision Xcite Multilabel plate reader</td>
			<td>PerkinElmer</td>
			<td>2104-0020A</td>
			<td>The assays can be performed on a variety of plate readers equipped with the TR-FRET option</td>
		</tr>
		<tr>
			<td>Erlotinib hydrochloride</td>
			<td>Sigma</td>
			<td>CDS022564</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tissue culture treated flasks</td>
			<td>Fisher</td>
			<td>13-680-65</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Wisent</td>
			<td>098-150</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa cell line</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>JAK Inhibitor 1</td>
			<td>Cayman Chemical</td>
			<td>15146</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital plate shaker</td>
			<td>Many options available</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human EGF</td>
			<td>PeproTech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IFN&#945;2b</td>
			<td>ProSpec</td>
			<td>CYT-460</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-4</td>
			<td>R&#38;D Systems</td>
			<td>204-IL</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute 1640 medium (RPMI)</td>
			<td>Wisent</td>
			<td>350-007-CL</td>
			<td>THUNDER TR-FRET is compatible with culture medium containing phenol red</td>
		</tr>
		<tr>
			<td>THUNDER Phospho-STAT1 (Y701) + Total STAT1 TR-FRET Cell Signaling Assay Kit</td>
			<td>BioAuxilium Research</td>
			<td>KIT-STAT1PT-500</td>
			<td></td>
		</tr>
		<tr>
			<td>THUNDER Phospho-STAT3 (Y705) + Total STAT3 Cell Signaling Assay Kit</td>
			<td>BioAuxilium Research</td>
			<td>KIT-STAT3PT-500</td>
			<td></td>
		</tr>
		<tr>
			<td>THUNDER Phospho-STAT4 (Y693) + Total STAT4 TR-FRET Cell Signaling Assay Kit</td>
			<td>BioAuxilium Research</td>
			<td>KIT-STAT4PT-500</td>
			<td></td>
		</tr>
		<tr>
			<td>THUNDER Phospho-STAT5 (Y694/Y699) + Total STAT5 TR-FRET Cell Signaling Assay Kit</td>
			<td>BioAuxilium Research</td>
			<td>KIT-STAT5PT-500</td>
			<td></td>
		</tr>
		<tr>
			<td>THUNDER Phospho-STAT6 (Y641) + Total STAT6 TR-FRET Cell Signaling Assay Kit</td>
			<td>BioAuxilium Research</td>
			<td>KIT-STAT6PT-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA 0.05%</td>
			<td>Wisent</td>
			<td>325-542-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>U266B1 cell line</td>
			<td>ATCC</td>
			<td>TIB-196</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>NA</td>
			<td>NA</td>
			<td>Use Milli-Q grade water (18 M&#937;.cm) to dilute Lysis Buffer and Detection Buffer</td>
		</tr>
	</tbody>
</table>

time-resolved f&#246;rster resonance energy transfer assays for measurement of endogenous phosphorylated stat proteins in human cells

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 F&#246;rster 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 &#181;L), 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.

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, <strong class="xfig"...

Watch this Scientific Journal Video about Time-resolved FÃ¶rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells at JoVE.com

Time-resolved F&amp;#246;rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

Time-resolved F&#246;rster 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.

Time-resolved F&#246;rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to ...

The TR-FRET based assays provide new cost effective tools for the screening and pharmacological characterization of specific and selective modulators of the JAK/STAT signaling pathways. This technique is much easier, rapid, reproducible, and robust than conventional methods like Western blot and ELISA. No washing steps are required and reagents are added in a single step.This immunoassay platform can be easily applied to other cell signaling proteins provided specific antibodies are available. To design reproducible assays, you must use good cell culture practices and establish standard operating procedures. In addition, you must carefully optimize the cell culture and treatment conditions.Demonstrating the procedure will be Genevieve Chatel, a scientist from our laboratory. To begin, culture HeLa and A431 cells in a humidified 37 degree Celsius and 5%carbon dioxide incubator using DMEM supplemented with 10%FBS. When the cells reach 70 to 80%confluence, trypsinize them and either passage or use them for assays.To perform the assay, dispense 50 microliters of cells at the pre-optimized density into a 96-well tissue culture-treated plate in the appropriate culture medium, then incubate them overnight in a 37 degree Celsius and 5%carbon dioxide incubator. The following day, prepare intermediate twofold and fourfold dilution series of the test compounds by serially diluting the compound in a serum-free medium across 12 wells of a polypropylene 96-well plate. For cell stimulation, add 50 microliters of serum-free medium containing the simulator at a 2X concentration and incubate the cells for the pre-optimized time at either room temperature or 37 degrees.For cell inhibition, add 25 microliters of serum-free medium containing the inhibitor at a 4X concentration and incubate the cells for the pre-optimized time at either room temperature or 37 degrees, then add 25 microliters of serum-free medium containing the stimulator at a 4X concentration and incubate for the pre-optimized time. Next, to lyse the cells, prepare the 1X supplemented lysis buffer and add phosphatase inhibitor cocktail to it. After carefully removing and discarding the cell culture medium, immediately add 50 microliters of the prepared 1X supplemented lysis buffer and incubate for 30 minutes at room temperature under shaking with moderate agitation.For TR-FRET detection, prepare the 4X antibody detection mix in 1X detection buffer, then prepare the EUAB1 and FRAB2 antibody solutions, as well as the EUAB3 and FRAB4 antibody solutions in 1X detection buffer. Finally, mix pre-diluted EUAB1 with pre-diluted FRAB2 for detection of phosphoprotein and pre-diluted EUAB3 and pre-diluted FRAB4 for detection of total protein. Then carefully pipette 15 microliters of the cell lysate from the 96-well culture plate to a well of a white low-volume 384-well microplate.Next, add 15 microliters of the positive control lysate and 15 microliters of 1X lysis buffer as a negative control to separate assay wells. To wells containing 15 microliters of the lysate, add five microliters of the corresponding 4X antibody detection mix to detect the phosphoprotein or total protein. After covering the plate with a plate sealer, incubate it at room temperature for one hour to overnight depending on the assay kit.After the incubation is complete, remove the adhesive plate sealer and read the plate on a TR-FRET compatible microplate reader. Results of TR-FRET assays for detection and quantification of phospho-STAT1 with total STAT1 and phospho-STAT4 in U266B1 cells along with phospho-STAT5 with total STAT5 in A431 cells are represented using concentration response curves. Similarly, TR-FRET assays for detection and quantification of phospho-STAT3 and total STAT3 and phospho-STAT6 and total STAT6 in HeLa cells are represented using concentration response curves.Overall, all assays showed robust TR-FRET signals, wide dynamic ranges, low inter-well coefficient variation, and acceptable signal-to-background ratios. Treatment of cells with JAK activators, interferon alfa-2B and IL-4 and EGF showed the anticipated concentration-dependent increase in STAT phosphorylation at specific tyrosine residues, while the corresponding total STAT proteins remained stable. Both JAK inhibitor and erlotinib inhibited the corresponding phospho-STAT levels in a concentration-dependent manner.Results of phospho-STAT4 stimulation by interferon alfa-2B in a suspension cell line or phospho-STAT6 stimulation by IL-4 in an adherence cell line using the one-plate protocol were consistent with those obtained using the two-plate protocol, thus showing successful adaptability of a two-plate transfer protocol to a one-plate all-in-one well protocol. Results of the intra-plate variability study for the phospho-STAT1 assay using a suspension cell line and the phospho-STAT3 assay using an adherence cell line demonstrate the robustness of these phospho-STAT assays for HTS applications. It is essential to supplement the lysis buffer with the phosphatase inhibitor cocktail to prevent dephosphorylation of the phosphorylated proteins from active phosphatase present in the whole cell extract.

time-resolved-forster-resonance-energy-transfer-assays-for

Research

JoVE Journal

Biochemistry

3.3K Views.  BioAuxilium Research. The TR-FRET based assays provide new cost effective tools for the screening and pharmacological characterization of specific and selective modulators of the JAK/STAT signaling pathways. This technique is much easier, rapid, reproducible, and robust than conventional methods like Western blot and ELISA. No washing steps are required and reagents are added in a single step.This immunoassay platform can be easily applied to other cell signaling proteins provided specific antibodies are av...