JAK3 (Ab-785) Antibody
Description
Mechanism of Action
JAK3 is a non-receptor tyrosine kinase critical for cytokine signaling through receptors sharing the interleukin-2 receptor gamma chain (γc). Phosphorylation at Tyr785 is a key regulatory site that enhances JAK3’s kinase activity. Studies demonstrate that phosphorylation at Tyr785 is essential for JAK3’s ability to phosphorylate downstream targets, such as STAT5 proteins, which mediate gene transcription .
Table 1: Key Features of JAK3 (Ab-785) Antibody
| Feature | Details |
|---|---|
| Target | Human, mouse, rat JAK3 (phosphorylated Tyr785) |
| Host | Rabbit |
| Clonality | Polyclonal |
| Applications | Western blotting (WB), ELISA, immunohistochemistry (IHC) |
| Immunogen | Synthetic peptide derived from human JAK3 (amino acids 751–800) |
Applications
The antibody is validated for multiple techniques:
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Western Blotting: Detects phosphorylated JAK3 in cell lysates (1:500–1:3000 dilution) .
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ELISA: Used to quantify JAK3 phosphorylation in serum or lysate samples .
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Immunohistochemistry: Localizes phosphorylated JAK3 in tissue sections (1:50–1:100 dilution) .
Role in Immune Signaling
Phosphorylation at Tyr785 is required for JAK3’s kinase activity and its interaction with cytokine receptors. Mutation of Tyr785 to phenylalanine (Y785F) reduces JAK3’s ability to phosphorylate STAT5 in vivo . This highlights Tyr785 as a critical site for JAK3 activation.
Disease Implications
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Immunodeficiency: JAK3 mutations, including Tyr785 phosphorylation defects, are linked to severe combined immunodeficiency (SCID) .
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Cancer: JAK3 signaling via Tyr785 phosphorylation promotes tumor cell migration and invasion in squamous cell carcinoma (SCCHN) .
JAK Inhibitors’ Impact
JAK inhibitors (e.g., baricitinib) reduce STAT3 phosphorylation downstream of JAK3, impairing B-cell activation and antibody production . This underscores JAK3’s role in adaptive immunity.
Product Specs
Target Background
- JAK3 mutations in Italian patients affected by SCID: New molecular aspects of a long-known gene. PMID: 30032486
- This study screened for mutations in SETBP1 and JAK3 in a cohort of seventy Italian patients with juvenile myelomonocytic leukemia, identifying 11.4% harboring secondary mutations in these genes and discovering two new mutations in the SKI domain of SETBP1. PMID: 26980750
- Jak3-mediated phosphorylation of beta-catenin suppressed EGF-mediated epithelial-mesenchymal transition and facilitated epithelial barrier functions by AJ localization of phosphorylated beta-catenin through its interactions with alpha-catenin. PMID: 28821617
- The frequency of JAK3 mutations in the JH2 domain was relatively low in extranodal natural killer/T-cell lymphoma, nasal type (NTCL), in contrast to a previous report. This study identified novel JAK3H583Y- and JAK3G589D-activating mutations that were oncogenic and sensitive to a JAK3 inhibitor. PMID: 28284718
- In natural killer/T-cell lymphoma (NKTL), phosphorylation of EZH2 by JAK3 promotes the dissociation of the PRC2 complex, leading to decreased global histone H3 lysine 27 methylation levels. PMID: 27297789
- JAK3 mediates smooth muscle cell proliferation and survival during injury-induced vascular remodeling. PMID: 28473442
- Data indicate that phosphorylation of Janus kinase 3 (JAK3) and STAT3 transcription factor (STAT3) was inhibited by latent membrane protein 1 (LMP1)-IgG. PMID: 28009988
- This study analyzed the JAK3 kinetic mechanism and inhibition by tofacitinib. PMID: 27555492
- A patient presented with a homozygous JAK3 mutation, while her parents were heterozygous carriers. PMID: 27593409
- JAK3 up-regulates SGLT1 activity by increasing the carrier protein abundance in the cell membrane, enhancing cellular glucose uptake into activated lymphocytes and contributing to the immune response. PMID: 27595398
- JAK3 and MCL-1 were down-regulated in patient CD8(+) T cells compared to their normal counterparts, likely due to defective suppressor activity of miR-29b and miR-198 in RCC CD8(+) T cells. PMID: 27063186
- N225K and A550V PTPN6 mutations cause loss-of-function leading to JAK3-mediated deregulation of the STAT3 pathway and reveal a mechanism that tumor cells can utilize to control PTPN6 substrate specificity. PMID: 26565811
- This study demonstrated that JAK3 mutations occur in 16% of patients with T-cell prolymphocytic leukemia, suggesting their potential significance as a prognostic marker. PMID: 26493028
- This study revealed that JAK3 is a powerful negative regulator of the creatine transporter SLC6A8. PMID: 26666525
- Case Report: Persistent rotavirus diarrhea post-transplant in a novel JAK3-SCID patient after vaccination. PMID: 26248889
- Interleukin-4 regulates hematopoietic lineage choice by activating the JAK3-STAT6 pathway, leading to dendritic-cell-specific DNA demethylation. PMID: 26829670
- Foxp3 exhibits a rapid turnover in Treg cells, partially controlled at the transcriptional level by the JAK/STAT pathway. PMID: 27077371
- Results confirm that JAK3 is mutated in T-PLL and highlight the potential therapeutic relevance of epigenetic regulators. PMID: 26917488
- Data provide the first evidence that deregulated Jak3/STAT3/STAT5 signaling in CTCL cells represses the expression of the gene encoding miR-22, a novel tumor suppressor miRNA. PMID: 26244872
- This study describes three patients with a novel deep intronic mis-splicing mutation in JAK3 as a cause of T-B+NK- severe combined immunodeficiency. PMID: 26769277
- Jak3 plays a role in promoting mucosal tolerance through suppressed expression and limiting activation of TLRs, preventing intestinal and systemic chronic low-grade inflammation, associated obesity, and metabolic syndrome. PMID: 26451047
- This study suggests that JAK3 may contribute to the pathogenesis of pediatric ALL and serve as a valuable therapeutic target for improving outcomes in pediatric patients with ALL. PMID: 25146434
- These results do not confirm an association between JAK3 polymorphisms and cardiovascular disease in rheumatoid arthritis. PMID: 25815310
- Activating Janus kinase 3 mutation is associated with mycosis fungoides. PMID: 26082451
- This study reports the first patient with relapsed pediatric Early T-cell precursor acute lymphoblastic leukemia to exhibit a homozygous JAK3 activating mutation, V674A, caused by acquired uniparental disomy. PMID: 25430085
- The JAK inhibitor ruxolitinib (that inhibits mainly the JAK3/STAT5 pathway) affects key characteristics of human NK cells, such as cytokine-induced expansion and killing via impaired cytokine-mediated NK cell activation. PMID: 25832652
- Case Report: JAK3 mutations giving rise to common variable immunodeficiency mimicking late-onset combined immunodeficiency. PMID: 26182690
- These results suggest that irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID: 25552479
- No JAK3 mutations (A572V or A573V) were identified in samples of NK cell neoplasms and EBV-associated T/NK cell lymphoproliferative disease in Japan. PMID: 23808814
- In extranodal natural killer/T cell lymphomas, phosphorylated JAK3 expression does not necessarily harbor exon 13 mutations. PMID: 24965108
- JAK3/STAT5 signaling via IL-2 receptor is necessary to maintain the long-term expression of the high-affinity alpha beta gamma(c)-receptor of IL-2 and optimal proliferation of blood lymphocytes. PMID: 25509109
- Report activated JAK3 variant in myelodysplastic syndromes. PMID: 24674452
- This study not only characterizes Jak3 interaction with Shc but also demonstrates the molecular mechanism of intracellular regulation of Jak3 activation. PMID: 24795043
- The finding of recurrent activating JAK3 mutations in patients with T-PLL could enable the use of JAK3 inhibitors to treat patients with this unfavorable malignancy who otherwise have a very poor prognosis. PMID: 24446122
- JAK3 is a powerful regulator of the peptide transporters PEPT1 and PEPT2. PMID: 23934551
- In cervical cancer cells, the phosphorylation of STAT5/JAK3 increases at low doses of IL-2 but significantly decreases at high doses. PMID: 24548303
- JAK3 mutations are found in a subset of clear cell renal cell carcinoma patients and may be associated with its development and a greater risk of metastases. PMID: 21868263
- This study highlights the essential role of Jak3 in the colon, where it facilitates mucosal differentiation by promoting the expression of differentiation markers and enhances colonic barrier functions through AJ localization of beta-catenin. PMID: 24045942
- Mutations of SETBP1 and JAK3 were common recurrent secondary events presumed to be involved in tumor progression and were associated with poor clinical outcomes. PMID: 23832011
- Hypomorphic Janus kinase 3 mutations result in a spectrum of immune defects, including partial maternal T-cell engraftment. PMID: 23384681
- This study analyzed the JAK-Fes-phospholipase D signaling pathway that is enhanced in highly proliferative breast cancer cells. PMID: 23404507
- Data suggest that cross-talk occurs between cAMP/PKA and the IL-2R beta/Jak3/Stat5b cascade in T-cells. PMID: 23341462
- Data indicate that the invasive phenotype of MDA-MB-231 cells is mediated by phospholipase D2 (PLD2) under direct regulation of both Janus kinase 3 (JAK3) and epidermal growth factor receptor (EGFR). PMID: 23238254
- This study analyzed the molecular mechanism of interactions between Jak3 and cytoskeletal proteins, where tyrosine phosphorylation of the SH2 domain acted as an intramolecular switch for the interactions between Jak3 and cytoskeletal proteins. PMID: 23012362
- Data suggest that Janus kinase 3 (JAK3) may play a significant role in the pathogenesis of natural killer/T-cell lymphoma (NKTCL). PMID: 22705984
- JAK3 mutations are associated with response to therapy in T-cell acute lymphoblastic leukemia. PMID: 22425895
- Peripheral blood lymphocytes (PBLs) of ovarian cancer patients showed lower JAK3, CD3-zeta molecules expression levels, as well as lower STAT3 and CD3-zeta phosphorylation levels than cells of control. PMID: 22221142
- This review analyzes the biochemistry, immunological functions, and clinical significance of JAK3. PMID: 22130498
- Data imply that IL-21-mediated signaling is critical for long-lived humoral immunity and to restore antibody responses in IL2RG/JAK3-deficient patients after hematopoietic cell transplantation. PMID: 22039266
- This review discusses the role of JAK3 inhibitors as immunosuppressive agents in kidney transplantation. PMID: 21971513
Q&A
What is the biological significance of JAK3 Tyr785 phosphorylation?
JAK3 (Janus kinase 3) undergoes phosphorylation at tyrosine 785 in response to IL-2 and other IL-2 family cytokines. This phosphorylation event is critically important for binding to SH2-Bβ, a signaling molecule in the cytokine cascade . Unlike the more extensively studied activation loop phosphorylation sites (Y980/Y981), Tyr785 represents a distinct regulatory mechanism in JAK3 function.
Methodological approach to study this significance:
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Stimulate cells expressing JAK3 with IL-2 or related cytokines
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Use phospho-specific antibodies to detect Y785 phosphorylation via Western blot
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Create Y785F mutants to assess functional consequences
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Compare timing of Y785 phosphorylation with other known phosphorylation events
How does JAK3 Tyr785 phosphorylation differ from other regulatory phosphorylation sites?
JAK3 contains multiple phosphorylation sites with distinct functions. Tyrosine 785 operates independently from the activation loop tyrosines (Y980/Y981). Research has shown that Y980F mutation and wild-type JAK3 display comparable levels of tyrosine phosphorylation, while mutations at Y904 and Y939 significantly reduce JAK3 kinase activity . The phosphorylation dynamics at Tyr785 appear to have distinct temporal and functional characteristics compared to these other sites.
| Phosphorylation Site | Effect on JAK3 Function | Response to IL-2 | Key Downstream Interactions |
|---|---|---|---|
| Tyr785 | Binding to SH2-Bβ | Rapidly induced | SH2-domain interactions |
| Tyr904/Tyr939 | Regulates kinase activity | Rapidly induced | ATP binding/substrate association |
| Tyr980/Tyr981 | Activation loop function | Rapidly induced | Catalytic activity |
What are the optimal protocols for detecting JAK3 Tyr785 phosphorylation in primary cells?
Detection of JAK3 phosphorylation at Tyr785 in primary cells requires careful optimization. Based on experimental data from multiple studies, the following protocol is recommended:
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Cell Stimulation: Stimulate primary human T cells with IL-2 (100 U/mL) or other IL-2 family cytokines for 5-15 minutes
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Cell Lysis: Use a lysis buffer containing phosphatase inhibitors (sodium orthovanadate, sodium fluoride, and β-glycerophosphate)
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Western Blot:
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Detection:
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Use HRP-conjugated secondary antibody and enhanced chemiluminescence
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Compare with total JAK3 detection in parallel samples
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How can phospho-flow cytometry be optimized for JAK3 (Tyr785) detection?
For researchers looking to implement phospho-flow cytometry for JAK3 Tyr785 detection:
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Cell Preparation:
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Stimulate cells with appropriate cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21)
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Fix cells immediately with 2% paraformaldehyde for 10 minutes at 37°C
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Permeabilize with ice-cold 90% methanol for 30 minutes
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Staining Protocol:
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Wash cells with PBS containing 0.5% BSA
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Incubate with anti-phospho-JAK3 (Tyr785) antibody (1:50 dilution) for 1 hour
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Include appropriate surface markers for identifying specific cell populations
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Analyze using standard flow cytometry techniques
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Similar approaches have been successful for other phospho-proteins in CyTOF analysis of immune cells .
How can JAK3 (Ab-785) antibody be used to evaluate the efficacy of JAK inhibitors?
JAK inhibitors represent an important class of therapeutics for autoimmune and inflammatory conditions. Using JAK3 (Ab-785) antibody to evaluate inhibitor efficacy requires:
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Experimental Design:
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Pre-treat cells with JAK inhibitors at various concentrations
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Stimulate with appropriate cytokines (e.g., IL-2 for JAK3 activation)
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Prepare cell lysates for Western blot or fixed cells for flow cytometry
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Detect both phospho-JAK3 (Tyr785) and total JAK3
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Comparative Analysis:
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Compare selective JAK1 inhibitors (e.g., filgotinib) and pan-JAK inhibitors (e.g., tofacitinib)
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Assess inhibition of downstream STAT phosphorylation
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Determine IC50 values for phospho-JAK3 inhibition
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Research indicates that unlike pan-JAK inhibitors, JAK1-specific inhibitors may not completely abrogate signaling due to continued activity of partnering JAKs . This has important implications for therapeutic development.
What experimental approaches can determine the functional consequences of JAK3 Tyr785 phosphorylation?
To establish the functional role of JAK3 Tyr785 phosphorylation:
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Mutagenesis Studies:
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Generate Y785F (phospho-null) and Y785E (phospho-mimetic) mutants
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Express in appropriate cell lines (e.g., JAK3-deficient cells)
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Compare with wild-type JAK3 expression
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Functional Readouts:
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Measure JAK3 kinase activity using in vitro kinase assays
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Assess STAT5 phosphorylation as a downstream marker
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Evaluate effects on cytokine-dependent proliferation and survival
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Monitor protein-protein interactions using co-immunoprecipitation
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Single-Cell Analysis:
What are common issues when using JAK3 (Ab-785) antibody and how can they be resolved?
Common challenges and solutions when working with JAK3 (Ab-785) antibody include:
How can researchers validate the specificity of JAK3 (Ab-785) antibody?
Proper validation of JAK3 (Ab-785) antibody specificity is critical for reliable research outcomes:
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Positive Controls:
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Specificity Tests:
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Peptide competition assay using the immunizing phosphopeptide
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JAK3 knockdown using siRNA or CRISPR
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JAK3-deficient cell lines as negative controls
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Cross-reactivity Assessment:
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Application-specific Validation:
How is JAK3 Tyr785 phosphorylation implicated in immune disorders?
JAK3 plays a crucial role in immune cell signaling, and dysregulation of its phosphorylation can contribute to various immune disorders:
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Experimental Approaches:
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Compare JAK3 Tyr785 phosphorylation levels in tissue samples from patients with autoimmune diseases versus healthy controls
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Analyze JAK3 phosphorylation in animal models of immune disorders
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Correlate phosphorylation levels with disease severity and therapeutic responses
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Research has shown that dysregulation of JAK-STAT signaling pathways contributes to autoimmune disorders, certain cancers, and immunodeficiencies . JAK3 mutations can result in severe combined immunodeficiency (SCID), highlighting its critical role in immune function.
What is the role of JAK3 in cancer progression and metastasis?
Recent studies have implicated JAK3 in tumor cell migration and invasion:
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Mechanistic Studies:
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Clinical Correlations:
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Compare JAK3 phosphorylation status with tumor stage and patient outcomes
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Assess JAK3 as a potential biomarker for metastatic potential
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Evaluate JAK3 inhibitors as potential anti-metastatic therapeutics
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Immunohistochemical analysis of patient samples can be performed using recommended protocols:
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Antigen retrieval with 10 mM sodium citrate buffer (pH 6.0)
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Blocking with normal goat serum
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Incubation with anti-phospho-JAK3 antibody (1:100 dilution) overnight at 4°C
This evidence suggests that JAK3 signaling may represent a therapeutic target in certain malignancies.
