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Pathway Focus: The JAK/STAT Pathway

Cell Staining Simulation tool

Primary Antibody Search Tool

3D Cell and Cell Staining Simulation tool iPad app

Imaging Global Transcription With Click-iT® RNA Assays

  • Rapid results—nascent RNA detection is complete typically within 30 minutes
  • Content-rich results—multiplex-compatible with other probes
  • Specificity—efficient incorporation by RNA polymerases but not DNA polymerases



Utilizing an alkyne-modified nucleoside, 5-ethynyl uridine (EU), and powerful click chemistry, newly synthesized RNA can be detected with a simple, two-step procedure. First, the alkyne-containing nucleoside is fed to cells or animals, and is actively and selectively incorporated into nascent RNA. Detection utilizes the chemoselective ligation or “click” reaction between an azide and an alkyne, where the modified RNA is detected with a corresponding azide-containing dye.

The JAK-STAT signaling pathway transmits information from chemical signals outside the cell to gene promoters in the nucleus, and affects cell proliferation, differentiation, and apoptosis.
Click-iT® RNA Alexa Fluor® Imaging Kits and HCS Assays provide the tools necessary to produce quantitative and qualitative measurements of RNA that arise from changes in DNA transcription.

The ability to detect newly synthesized RNA or changes in RNA levels will aid in understanding viral gene expression and genome replication, as well as its subcellular localization in host cells. The small size of the Click-iT® detection molecule allows easy penetration of complex samples. The assay is multiplex-compatible with other probes, including antibodies, for the simultaneous detection of RNA-interacting proteins.

Dose response for actinomycin D in HeLa cells using the Click-iT® RNA HCS Assay
Dose response for actinomycin D in HeLa cells using the Click-iT® RNA HCS Assay.

HeLa cells were treated with the indicated amounts of actinomycin D for 18 hr, followed by a 1 hr incubation with 5-ethynyl uridine (EU). Cells were then fixed and permeabilized, and EU incorporated into newly synthesized RNA was detected using green-fluorescent Alexa Fluor® 488 azide. Quantitative analysis was performed using the Cellomics® ArrayScan® VTI and Compartmental Analysis Bioapplication (Thermo Scientific).
For research use only. Not intended for any animal or human therapeutic or diagnostic use.
Click-iT® RNA Assay Selection Guide

Description
Cat. No.
Click-iT® RNA Alexa Fluor® 488 HCS Assay, 2-plate size
C10327
Click-iT® RNA Alexa Fluor® 594 HCS Assay, 2-plate size
C10328
Click-iT® RNA Alexa Fluor® 488 Imaging Kit, for 25 coverslips
C10329
Click-iT® RNA Alexa Fluor® 488 Imaging Kit, for 25 coverslips
C10330

Gibco® Cytokines and Growth Factors that Stimulate the JAK/STAT Pathway

  • High biological activity
  • High purity
  • Proven compatibility with Gibco® media

 



Gibco® Cytokines and Growth Factors have been bioassayed with Gibco® media to help ensure compatibility, making them useful in a wide variety of applications, including cell stimulation, proliferation, and differentiation.

STAT proteins are a family of cytoplasmic signaling proteins involved in signal transduction of several cytokines and growth factors.

In response to the cytokine or growth factor binding to cell surface receptors, STAT proteins become activated by tyrosine phosphorylation. Phosphorylated STAT proteins subsequently move to the cell nucleus, where they activate transcription.

Individual cytokines are thought to activate specific STAT family members. For example, STAT1 and STAT2 play a central role in the interferon signaling cascade. The binding of interleukin 6 (IL-6) family cytokines and some other growth factors triggers STAT3 phosphorylation.

IL-2, IL-3, IL-5, IL-7, IL-15, GM-CSF, thrombopoietin, and different growth hormones activate the STAT5 protein.

Gibco® Cytokines and Growth Factors are high-purity recombinant proteins with high bioactivity. To ensure Gibco® growth factors are of the highest quality, each protein is analyzed for purity and for structural homogeneity to help ensure that a biologically active protein is produced.

For research use only. Not intended for any animal or human therapeutic or diagnostic use.
Gibco® Cytokine and Growth Factor Selection Guide

Product Cat. No.
Product
Cat. No. Product
Cat. No.
CNTF
PHC7015IL-10
PHC0104
IL-4
PHC0044
EGF
PHG0314IL-12
PHC1124
IL-5
PHC0055
G-CSF
PHC2035IL-13
PHC0134IL-6
PHC0064
GM-CSF
PHC2015IL-15
PHC9154IL-7
PHC0075
IFN-alpha
PHC4814IL-2
PHC0026PDGF
PHG0044
IFN-beta
PHC4244IL-20PHC0204TPO
PHC9514
IFN-gamma
PHC4031IL-3
PHC0034

Multiplex STAT Luminex® Assay—Simultaneously Measure STAT1, STAT3, and STAT5a/b

  • Superior performance—enables accurate quantitation of the STAT pathway proteins
  • High quality—in-house manufactured antibodies allow for high specificity and sensitivity
  • Fast and easy—collect and analyze your data typically in less than one day



These 3-plex Luminex® assays provide a series of combined reagents for the simultaneous measurement of phosphorylated and total STAT1, STAT3, and STAT5a/b in cell lysates and tissue homogenates.

STAT proteins control transcription of specific genes in response to cytokine stimulation. Given that the diverse cellular response to IFN exposure is mediated by a common pathway, it is likely that a variety of STAT homo- and heterodimer combinations are responsible for each individual phenotype through differential gene regulation.
Differential activation of STAT1, STAT3, and STAT5a/b in different cell types suggests the possibility of different STAT homo- and heterodimer populations in response to interferon treatment. Significant activation of both STAT1 and STAT3 in TF-1 cells in response to IFN-α suggests a greater population of STAT1/STAT3 heterodimers. This is in contrast to TF-1 cell response to IFN-γ, where only STAT1 is significantly activated and STAT1 homodimers are the most likely transcriptional regulators.

This figure shows the advantages inherent to multiplex analysis for activation effects of cytokines. The ability to monitor activation of STAT1, STAT3, and STAT5a/b in a single well provides advantages in terms of time and sample requirements, and allows different markers in the same sample to be compared.
STAT 1, 3, and 5 phosphorylation measured in one sample using the Luminex® bead-based platform for multiplexing capabilities
STAT 1, 3, and 5 phosphorylation measured in one sample using the Luminex® bead-based platform for multiplexing capabilities.

STAT 1, 3, and 5 phosphorylation measured in one sample using the Luminex® bead-based platform for multiplexing capabilities. TF-1 cells were serum starved overnight, then stimulated with specific cytokines for 15 minutes. Untreated cells were used as an unstimulated control. Measured levels of STAT proteins were as expected following specific treatments using the STAT 1, 3, 5 Plex Antibody Bead Kit.
For research use only. Not intended for any animal or human therapeutic or diagnostic use.
Multiplex STAT Luminex® Assay Selection Guide

Product
Cat. No.
STAT1, 3, 5a/b Phospho 3-Plex Panel
LHO0005
STAT1, 3, 5a/b Total 3-Plex Panel
LHO0791

CellTrace™ Violet Cell Proliferation Assay—Violet Laser Cell Proliferation Reagents Make Analysis in GFP-Expressing Cells Possible

  • Versatility—violet laser excitation enables multiplex detection with other commonly used cellular labels such as FITC and GFP
  • Superior performance—bright, narrow emission peaks enable simplified visualization of multiple generations
  • Long-term signal stability—retained in cells for several days post-stain
  • Noncytotoxic effects—minimal effect on proliferative ability or biology of cells

Like CFSE, the new Molecular Probes® CellTrace™ Violet Cell Proliferation Kit allows quantitation of cell generations by dye dilution using flow cytometry. Now with the violet excitation and blue emission of CellTrace™ Violet, you can perform proliferation experiments with a wide range of blue-excitable fluorophores, including GFP.

Stained human lymphocytes
Stained human lymphocytes.
Human CD8+ T lymphocytes stained with 10 µM CellTrace™ Violet followed by incubation in OpTmizer™ T Cell Expansion Medium at 37°C for 7 days. Cells were stimulated with 200 ng mouse anti–human CD3 antibody and 100 ng interleukin-2 per milliliter of cells.
For research use only. Not intended for any animal or human therapeutic or diagnostic use.
Order the CellTrace™ Violet Cell Proliferation Kit

Description
Laser
Ex/Em
Cat. No.
CellTrace™ Violet Cell Proliferation Kit
405 nm
405/450
V34557

Better Sensitivity Measuring JAK/STAT Proteins—phosphoELISA™ Kits

  • More sensitive than western blots
  • Small volume of cell lysate needed
  • Quantitative results typically after only 4 hours of incubation time 


The JAK/STAT signaling pathway plays an important role in regulating cell proliferation, differentiation, and apoptosis. Upon binding of a variety of cytokines and growth factors to the appropriate receptors, JAK kinases are recruited and activated. JAKs then phosphorylate the receptors’ cytoplasmic domain, causing recruitment of STATs, which are in turn phosphorylated, dimerized, and translocated into the nucleus. Once in the nucleus, STAT family members (STAT1, 2, 3, 4, 5a, 5b, and 6) control transcription of specific genes in response to stimulation.

phosphoELISA™ kits allow you to measure the levels of total and phosphorylated JAK and STAT proteins with quantitative results in only half a day.  phosphoELISA™ kits provide a simple and unbiased way to quantify JAK/STAT protein levels with high specificity and better sensitivity than western blots. The kits come ready to use with all the necessary reagents, including recombinant standards that allow quantitative measurements. In addition, the flexible 96-strip well plate format allows you to run as many samples, or as few, as you need.

Cell extracts were prepared and analyzed with the STAT5a [pY694] ELISA and STAT5a (Total) ELISA kits
Cell extracts were prepared and analyzed with the STAT5a [pY694] ELISA and STAT5a (Total) ELISA kits.
Phosphorylation of STAT5a is increased in sodium vanadate–treated HEL cells, whereas the total level of STAT5a remains relatively constant in treated vs. untreated control, demonstrating the utility of the Total ELISA kits as controls.
JAK/STAT phosphoELISA™ Kit Selection Guide

Description
Species
Quantity
Cat. No.
STAT3 [pY705] ELISA Kit
Hu, Ms, Rt
96 tests
KHO0481
STAT5a [pY694] ELISA Kit
Hu96 tests
KHO0761
STAT5a (Total) ELISA Kit
Hu96 tests
KHO0751
STAT5b [pY699] ELISA Kit
Hu96 tests
KHO5721
STAT6 [pY641] ELISA Kit
Hu96 tests
KHO0801
JAK2 [pYpY1007/1008] ELISA Kit
Hu96 tests
KHO5621
JAK2 (Total) ELISA Kit
Hu96 tests
KHO5521

Antibodies for JAK/STAT Signaling Pathways

  • Selection—over 700 different phosphorylation site–specific antibodies
  • Validation—effective in a range of applications, including IHC and flow cytometry
  • Specificity—ABfinity™ technology helps ensure quality


We offer over 700 phosphorylation site–specific antibodies, and antibodies against extracellular markers to study the JAK/STAT and related pathways. This includes phosphorylation site–specific antibodies, which are tested on western blots with phosphorylation site–blocking peptides to ensure the phosphorylation specificity.

The JAK/STAT pathway is one of the crucial signaling pathways affecting cell functions, including proliferation, growth, hematopoiesis, and immune response. Our high-quality antibodies are ideal for experiments to detect total protein and even activated fractions. This information is valuable as scientist work toward understanding the role of each pathway protein.

Our JAK/STAT antibodies are validated not only in traditional techniques like western blotting and ELISA, but also in more challenging techniques like immunofluorescence, immunohistochemistry, and flow cytometry.

The availability of JAK/STAT antibodies produced with ABfinity™ recombinant rabbit monoclonal antibody technology enables researchers to have another layer of assurance in the quality and lot-to-lot consistency of these antibodies (STAT4 ABfinity™ Recombinant Rabbit Monoclonal Antibody and SUMO-3 ABfinity™ Recombinant Rabbit Monoclonal Antibody).

Order ABfinity™ Recombinant Rabbit Monoclonal Antibodies

Description
Cat. No.
STAT4 ABfinity™ Recombinant Rabbit Monoclonal Antibody
700185
SUMO-3 ABfinity™ Recombinant Rabbit Monoclonal Antibody
700186