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Pathway Focus: The JAK/STAT Pathway
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Multiplex STAT Assay—Simultaneously Measure STAT1, STAT3, and STAT5a/b Activations
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. |
) (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. |
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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. |
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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. |
| Product | Premixed Panel | Species | Cat. No. | |
| STAT1, 3, 5a/b Phospho 3-Plex Panel | STAT1 [pY701], STAT3 [pY705], and STAT5a/b [pY694/699] | Hu | LHO0005 |  |
| STAT1, 3, 5a/b Phospho 3-Plex Panel | STAT1, STAT3, and STAT5a/b | Hu | LHO0791 | |
Measuring JAK/STAT Proteins Using phosphoELISA™ Kits—Quickly Detect and Quantify Phosphorylation Site-Specific Proteins
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). |
 | 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. |
 | 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 Kit | Hu, Ms, Rt | 96 tests | KHO0481 | |
| STAT5a [pY694] ELISA Kit | Hu | 96 tests | KHO0761 | |
| STAT5a (Total) ELISA Kit | Hu | 96 tests | KHO0751 | |
| STAT5b [pY699] ELISA Kit | Hu | 96 tests | KHO0761 | |
| STAT6 [pY641] ELISA Kit | Hu | 96 tests | KHO0801 | |
| JAK2 [pYpY1007/1008] ELISA Kit | Hu | 96 tests | KHO5621 | |
| JAK2 (Total) ELISA Kit | Hu | 96 tests | KHO5521 | |
Recombinant Proteins to Induce JAK/STAT Signaling—High Quality GIBCO® Recombinant Proteins
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. |
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| Description | Species | Quantity | Cat. No. | |
| IFN-α A Pure Recombinant Human | Hu | 5µg | PHC4014 | |
| IFN-α A Pure Recombinant Mouse | Ms | 1 x 105 units | PMC4016 | |
- View Full List of Recombinant Proteins for JAK/STAT Signaling
Antibodies for JAK/STAT Signaling Pathways—Phospho-Antibodies for Pathway Analysis
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. |
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| Description | Cat. No. | |
| STAT4 ABfinity™ Recombinant Rabbit Monoclonal Antibody | 700185 | |
| SUMO-3 ABfinity™ Recombinant Rabbit Monoclonal Antibody | 700186 | |
| FIX & PERM® | GAS004 | |
| Alexa Fluor® 488 goat anti-rabbit Ig | A11008 | |
| SuperPicTure™ Polymer DAB | 87-8963 | |
