BioPath Online

Pathway Focus: The JAK/STAT Pathway

Multiplex STAT assay
Multiplex STAT Assay—Simultaneously Measure STAT1, STAT3, and STAT5a/b Activations
Measuring JAK/STAT Proteins Using phosphoELISA™ KitsMeasuring JAK/STAT Proteins Using phosphoELISA™ Kits—Quickly Detect and Quantify Phosphorylation Site-Specific Proteins
Recombinant Proteins to Induce JAK/STAT SignalingRecombinant Proteins to Induce JAK/STAT Signaling—High Quality GIBCO® Recombinant Proteins
Antibodies for JAK/STAT Signaling PathwaysAntibodies for JAK/STAT Signaling Pathways—Phospho Antibodies for Pathway Analysis
New Antibodies

New Immunoassays

New Molecular Probes® Products

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

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


IFNs are classified according to cellular origin and extracellular receptor binding specificity. Type I IFNs include IFN-α and IFN-β. IFN receptor interactions with both IFNs lead to activation of intracellular kinases which transmit signals to the cell nucleus through a series of phosphorylation events. The best-characterized and commonly accepted IFN signaling pathway involves activating members of the Janus kinase (JAK) family upon IFN binding; JAKs then phosphorylate substrate proteins known as signal transducers and activators of transcription (STATs). The STAT family is comprised of STATs 1, 2, 3, 4, 5A, 5B, and 6.

STATs control transcription of specific genes in response to cytokine stimulation. Events leading to STAT activation arising from IFN stimulation are illustrated in Figure 1. 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.

We quantified the activation of three STAT family members (STAT1, STAT3, and STAT5) in a variety of cells exposed to IFN-α, IFN-β, or IFN-γ, using both individual ELISAs and a multiplex-bead immunoassay developed for simultaneous detection of activated proteins (Figure 2).
All three cell types showed significant activation of STAT1 by IFN-α and IFN-β. These Type I interferons also activated STAT3 in Jurkat and TF-1 cells. Cellular response to IFN-γ treatment was mostly limited to STAT1 activation in Jurkat and TF-1 cells. Levels of STAT5a/b activation remained low in all cell types and treatment conditions, with the exception of IFN-α treatment of TF-1 cells. Activation patterns by cell type were similar for IFN-α and IFN-β, consistent with them both binding to the same type I cell surface receptor. Distinct activation patterns for IFN-γ suggest binding to its own type II receptor. The pattern of STAT activation was distinct for each different cell type.

Differential activation of STAT1, STAT3, and STAT5a/b in different cell types suggest the possibility of different STAT homo- and hetero-dimer populations in response to interferon treatment. Significant activation of both STAT1 and STAT3 in TF-1 cells in response to IFN-α suggest a greater population of STAT1/STAT3 hetero-dimers (Figure 3). This is in contrast to TF-1 cell response to IFN-γ, where only STAT1 is significantly activated and STAT1 homo-dimers are the most likely transcriptional regulators.

These results show the advantages inherent to a 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, sample requirements, and comparison of different markers in the same sample.

 

Events leading to STAT activation
(click to enlarge)
 Figure 1. Events leading to STAT activation. (A) Extracellular IFN (blue triangle) prior to binding to its receptor. (B) Upon binding, JAK kinases constitutively associated with the receptor subunits interact. The interacting JAK proteins activate one another by reciprocal tyrosine phosphorylation, and phosphorylate a tyrosine on two subunits contained in the receptor complex. These phosphorylated tyrosine residues provide paired docking sites for STAT via its SH2 domain (C). STAT, recruited to the receptor complex, is then phosphorylated at a tyrosine residue by the JAKs. This tyrosine phosphorylation promotes STAT homo and heterodimerization mediated by reciprocal phosphotyrosine SH2 domain interaction and the dissociation of the dimers from the receptor complex (D). The STAT dimer is then translocated to the cell nucleus, where they complex with other nuclear proteins and regulate gene expression by binding to promotors or other response elements on DNA.

 

Differential STAT activation by cell type and IFN treatment
(click to enlarge)

Figure 2. Differential STAT activation by cell type and IFN treatment. Phospho-specific multiplex bead immunoassay quantitation of STAT1, STAT3 and STAT5a/b activation in 4 different cell types. Cells were serum starved overnight and treated as indicated in the figure. Untreated cells were used as controls. Cell extracts were prepared from each treatment and 400 μg/mL of each cell extract was analyzed using a single multiplex bead immunoassay.

 

Treatment with IFN-α and IFN-γ

Figure 3. Treatment with IFN-α and IFN-γ show the highest degree of STAT activation. Phospho-specific multiplex bead immunoassay data re-plotted to illustrate patterns of STAT activation in each cell type treated with either IFN-α or IFN-γ.

 

ProductPremixed PanelSpecies Cat. No. 
STAT1, 3, 5a/b Phospho 3-Plex PanelSTAT1 [pY701], STAT3 [pY705], and STAT5a/b [pY694/699]HuLHO0005
STAT1, 3, 5a/b Phospho 3-Plex PanelSTAT1, STAT3, and STAT5a/bHuLHO0791

 

Measuring JAK/STAT Proteins Using phosphoELISA™ Kits—Quickly Detect and Quantify Phosphorylation Site-Specific Proteins

  • Better understand JAK or STAT protein recruitment
  • Measure total and phosophorylation site–specific JAK/STAT proteins
  • Quantify in just 4 hr

JAK and STAT are signaling proteins. Upon binding of a variety of cytokines and growth factors to the appropriate receptor, JAK kinases are recruited and activated. JAKs then phosphorylate the receptor’s cytoplasmic domain, causing recruitment of STATs, which are in turn phosphorylated, dimerized, and translocated into the nucleus. At that point, STAT family members (STAT1, 2, 3, 4, 5a, 5b, and 6) control transcription of specific genes in response to stimulation.

Although the basic mechanism of the JAK/STAT pathway is known, very little is known about the details of the recruitment of particular JAK or STAT proteins. For example, cytokine receptors demonstrate preferential use of either a single JAK or a JAK combination.
STATs, on the other hand, exhibit either cell type–specific induction of transcription or stereotypic transcription, regardless of cell type. Current research has focused on identifying JAK/STAT family inhibitors for areas of oncology and immune suppression. 

Invitrogen phosphoELISA™ kits allow you to measure the levels of total and phosphorylation site–specific JAK and STAT proteins (Figure 4). phosphoELISA™is a simple and unbiased way to quantify JAK/STAT protein levels with high specificity and sensitivity in only 4 hours. The kits come ready to use with all necessary reagents, including recombinant standards for quantitative results.  In addition, the flexible 96-stripwell plate format allows you to run as many samples, or as few, as you need (Figure 5).

 

Total ELISA kits make effective controls Figure 4. Total ELISA kits make effective controls. 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.

 

ELISA kits comparable to westerns Figure 5. ELISA kits comparable to westerns. In parallel, cell extracts were prepared and analyzed in western blots and phosphoELISA™ kits for STAT5 [pY694].  Band intensity in the western blot corresponds to quantitative O.D. results from the ELISA kit.

 

Description
Species
Qty.Cat. No. 
STAT3 [pY705] ELISA KitHu, Ms, Rt96 testsKHO0481
STAT5a [pY694] ELISA KitHu96 testsKHO0761
STAT5a (Total) ELISA KitHu96 testsKHO0751
STAT5b [pY699] ELISA KitHu96 testsKHO0761
STAT6 [pY641] ELISA KitHu96 testsKHO0801
JAK2 [pYpY1007/1008] ELISA KitHu96 testsKHO5621
JAK2 (Total) ELISA KitHu96 testsKHO5521

 

Recombinant Proteins to Induce JAK/STAT Signaling—High Quality GIBCO® Recombinant Proteins

  • Pure, bioactive proteins to induce signaling
  • High biological activity
  • Compatible with media systems


JAK/STAT pathways are activated by a variety of cytokines and growth factors. Upon ligand binding to the receptor, JAK is activated, followed by phosphorylation of STATs on specific tyrosine residues. Invitrogen™ pure bioactive recombinant proteins can induce JAK/STAT signaling. In figure 6, TF-1 cells were treated with IFN-α or IL-3, or left unstimulated. Cell lysates were analyzed using the STAT5b [pY699] ELISA kit. Treatment with IFN-α or IL-3 results in activation of the JAK/STAT pathway, as seen by the upregulation of phosphorylated STAT5b.
Expression of STAT5b (pY699) in TF-1 cells

Figure 6. Expression of STAT5b (pY699) in TF-1 cells.



DescriptionSpecies
Quantity Cat. No.

IFN-α A Pure Recombinant Human
Hu
5µg
PHC4014
Order 
IFN-α A Pure Recombinant Mouse
Ms
1 x 105 units
PMC4016Order

Antibodies for JAK/STAT Signaling Pathways—Phospho-Antibodies for Pathway Analysis

  • Widest portfolio—including phosphor-site-specific antibodies
  • Specific—each lot tested with phospho-site peptide blocking western blot
  • Top quality and consistency—the new ABfinity™ next-generation antibodies


The JAK/STAT signaling pathway consists of around four different JAKs and different seven STATs, in addition to cytokines and other molecules. We offer one of the best portfolios for most of these targets, including phospho-site–specific antibodies that detect the protein only at a specific phospho-site. To make sure Invitrogen antibodies detect only that site, each lot is tested with phopho-site peptide blocking western blot. These antibodies are also validated against a variety of species and with most applications. 

To increase lot-to-lot consistency and quality of antibodies, Invitrogen is proud to introduce the next generation of antibodies:  ABfinity™ antibodies. These antibodies are produced by immunizing rabbits. Genes for those highly specific antibodies are cloned and expressed in a mammalian expression system.
Since each lot is produced by transfecting mammalian cells with known DNA, there are almost no variables that can affect antibody quality. For JAK/STAT pathway research, we introduce the STAT4 ABfinity™ Recombinant Rabbit Monoclonal Antibody, and SUMO-3 ABfinity™ Recombinant Rabbit Monoclonal Antibody (700186). 

High-quality Invitrogen™ antibodies can help researchers detect not only the total amount of each type of protein in certain cells or cell types, but also determine how much protein is activated at specific cites. This helps aid in understanding the role of each pathway member in the root causes of different diseases.

Invitrogen offers over 700 phospho-site–specific antibodies and antibodies against extracellular markers to study JAK/STAT and related pathways. These antibodies are not only validated in traditional techniques like western blot and ELISA, but also for more advanced techniques like immunofluorescence, immunohistochemistry, and flow cytometry (figures 7–10). The availability of ABfinity™ antibodies give researchers another layer of assurance due to the quality and lot-to-lot consistency of these antibodies.

 

Flow cytometry of Jurkat cells labeled with rabbit anti-STAT4

Figure 7. Flow cytometry of Jurkat cells labeled with rabbit anti-STAT4. Jurkat cells were fixed and permeabilized using FIX & PERM® reagents. Cells were stained with (black trace) or without (blue trace) 0.5 μg anti-STAT4 followed by Alexa Fluor® 488 goat anti-rabbit Ig. Pre-incubation with  immunogenic peptide decreased the signal (red trace).
 
Immunohistochemistry of human normal testes and colon carcimona tissue(click to enlarge)

Figure 9. Immunohistochemistry of human normal testes and colon carcinoma tissue labeled with rabbit anti-STAT4. FFPE human normal testes (left) and colon carcinoma (right) tissues were labeled with rabbit anti-STAT4 (5 µg/ml). Tissues were pretreated with EDTA and detected with SuperPicTure™ Polymer DAB. Images were taken at 40x magnification. Note cytoplasmic and nuclear staining in spermatocytes (left) and tumor cells (right).
 
Immunocytochemistry of A549 cells labeled with rabbit anti-STAT4(click to enlarge)

Figure 8. Immunocytochemistry of A549 cells labeled with rabbit anti-STAT4. A549 cells labeled with rabbit anti-STAT4 (2.5 µg/ml) in the absence of peptides (left) or in the presence of peptide used as immunogen (right). Alexa Fluor® 488 goat anti-rabbit at 1:1,000 was used as secondary antibody.
 
Western blot of multiple lysates

Figure 10. Western blot of multiple lysates labeled with rabbit anti-STAT4. Rabbit anti-STAT4 (3 µg/ml) was used to label STAT4 in mouse testes (lane 1), rat testes (lane 2), and A549 cells (lane 3).

 

DescriptionCat. No. 
STAT4 ABfinity™ Recombinant Rabbit Monoclonal Antibody700185Order 
SUMO-3 ABfinity™ Recombinant Rabbit Monoclonal Antibody700186Order
FIX & PERM®GAS004Order
Alexa Fluor® 488 goat anti-rabbit IgA11008Order
SuperPicTure™ Polymer DAB87-8963Order