Phospho-JUN (Y170) Antibody
Description
Overview of Phospho-JUN (Y170) Antibody
Phospho-JUN (Y170) Antibody (e.g., A27334 ) is a rabbit polyclonal antibody designed to specifically recognize c-Jun phosphorylated at tyrosine residue 170 (Y170). This post-translational modification regulates c-Jun’s stability, interactions with ubiquitin ligases (e.g., Itch), and downstream transcriptional activity .
Development and Specificity
-
Immunogen: Synthetic phosphopeptide derived from the Y170 region of human c-Jun .
-
Host Species: Rabbit.
-
Cross-Reactivity: Validated for human, mouse, and rat samples .
-
Specificity: Binds exclusively to phosphorylated Y170, confirmed via knockout/knockdown controls and peptide competition assays .
Applications in Research
This antibody is widely used in:
-
Western Blot (WB): Detects phosphorylated c-Jun (~37 kDa) in lysates from stress-treated cells .
-
Immunohistochemistry (IHC): Localizes Y170-phosphorylated c-Jun in paraffin-embedded tissues .
Role of Y170 Phosphorylation in c-Jun Regulation
Conflicting Observations
-
While Csk and c-Abl kinases phosphorylate Y170 , mutagenesis studies show Y170 is dispensable for c-Jun turnover .
-
Y170 phosphorylation does not impair JNK-mediated phosphorylation at S63/S73 .
Limitations and Considerations
Product Specs
Target Background
- Research has shown that miR-139-5p is down-regulated in the hearts of Hypertrophic cardiomyopathy patients and that it inhibits cardiac hypertrophy by targeting c-Jun expression. PMID: 29440459
- This study identified a crucial Jun/miR-22/HuR regulatory axis in CRC (the working model is summarized in Fig. 8) and highlighted the vital role of HuR and miR-22 in CRC proliferation and migration. PMID: 29351796
- A novel cascade mediated by AP-1 and FOXF1 that regulates oncogene-induced senescence is reported. PMID: 30119690
- Multivalent interactions with Fbw7 and Pin1 facilitate recognition of c-Jun by the Fbw7. PMID: 29225075
- High AP-1 expression is associated with metastasis in colon cancer. PMID: 29305742
- Our findings suggest that extended AP-1 binding sites, together with adjacent binding sites for additional TFs, encode part of the information that governs transcription factor binding sites activity in the genome. PMID: 29305491
- The expression of WIF-1 was low in GBC cells due to aberrant hypermethylation of its promoter region. Additionally, an alternative pathogenesis of GBC was indicated in which c-Jun causes hypermethylation of the WIF-1 promoter region, and represses the expression of WIF-1 through transcriptional regulation and interaction with DNMT1 as an early event in the tumorigenesis of GBC. PMID: 29693707
- Mutant cellular AP-1 proteins promote expression of a subset of Epstein-Barr virus late genes in the absence of lytic viral DNA replication. PMID: 30021895
- Secreted Ta9 has, therefore, not only the ability to stimulate CD8+ T cells but also the potential to activate AP-1-driven transcription and contribute to T. annulata-induced leukocyte transformation. PMID: 29738531
- MiR-216b directly targets c-Jun, thereby reducing AP-1-dependent transcription and sensitizing cells to ER stress-dependent apoptosis. PMID: 27173017
- Results suggest that c-Jun, p38 MAPK, PIK3CA/Akt, and GSK3 signaling are involved in the effect of miR-203 on the proliferation of hepatocellular carcinoma cells. PMID: 28887744
- These findings suggest that increased JUN expression and activity may contribute to gefitinib resistance in non-small cell lung cancer. PMID: 28566434
- The results indicated that butein has antiproliferative and proapoptotic properties through the suppression of NF-kappaB, AP-1, and Akt signaling in HTLV-1-infected T cells, both in vitro and in vivo, suggesting its therapeutic potential against HTLV-1-associated diseases including adult T-cell leukemia/lymphoma. PMID: 28586006
- Results show that VEGFA induces c-jun expression in mediating human retinal microvascular endothelial cell migration, sprouting, and tube formation, and that Pyk2-STAT3 signaling enhances cJun expression in the mediation of retinal neovascularization. PMID: 27210483
- Increased c-jun expression is associated with nasopharyngeal carcinoma. PMID: 28269757
- Thrombin binding to PAR-1 receptor activated Gi-protein/c-Src/Pyk2/EGFR/PI3K/Akt/p42/p44 MAPK cascade, which in turn elicited AP-1 activation and ultimately evoked MMP-9 expression and cell migration in SK-N-SH cells. PMID: 27181591
- Findings provide evidence that phospho-c-Jun activates an important regulatory mechanism to control DNMT1 expression and regulate global DNA methylation in glioblastoma. PMID: 28036297
- Results demonstrated for the first time the regulatory mechanism of miR-744 transcription by c-Jun, providing a potential mechanism underlying the upregulation of miR-744 in cancers. PMID: 27533465
- Results provide evidence that NuRD represses c-Jun transcription directly which, in the absence of MBD3, activates endogenous pluripotent genes and regulates induced cancer stem cells-related genes. PMID: 27894081
- Taken together, these results indicated that PAR1 signaling-mediated cJun activation promotes early apoptosis of HUVEC cells induced by heat stress. PMID: 28447716
- Cheliensisin A (Chel A) treatment led to PH domain and Leucine rich repeat Protein Phosphatases (PHLPP2) protein degradation and subsequently increased in c-Jun phosphorylation, which could be attenuated by inhibition of autophagy mediated by Beclin 1. PMID: 27556506
- The positive feedback regulation of OCT4 and c-JUN, resulting in the continuous expression of oncogenes such as c-JUN, seems to play a critical role in the determination of the cell fate decision from induced pluripotent stem cells to cancer stem cells in liver cancer. PMID: 27341307
- miR-26b plays an anti-metastatic role and is downregulated in gastric cancer tissues via the KPNA2/c-jun pathway. PMID: 27078844
- The IL1B/AP-1/miR-30a/ADAMTS-5 axis regulates cartilage matrix degradation in osteoarthritis. PMID: 27067395
- TGM2 is involved in amyloid-beta (1-42)-induced pro-inflammatory activation via AP1/JNK signaling pathways in cultured monocytes. PMID: 27864692
- Integrative genomic analysis indicated overexpression of the AP-1 transcriptional complex suggesting experimental therapeutic rationales, including blockade of the renin-angiotensin system. This led to the repurposing of the angiotensin II receptor antagonist, irbesartan, as an anticancer therapy, resulting in the patient experiencing a dramatic and durable response. PMID: 27022066
- Knockdown of CD44 reduced the protein level of xCT, a cystine transporter, and increased oxidative stress. However, an increase in GSH was also observed and was associated with enhanced chemoresistance in CD44-knockdown cells. Increased GSH was mediated by the Nrf2/AP-1-induced upregulation of GCLC, a subunit of the enzyme catalyzing GSH synthesis. PMID: 28185919
- This study highlights the role of AP1 in promoting the host gene expression profile that defines Ebola virus pathogenesis. PMID: 28931675
- This is the first study to show how TGF-beta regulates the expression of Claudin-4 through c-Jun signaling and how this pathway contributes to the migratory and tumorigenic phenotype of lung tumor cells. PMID: 27424491
- Data show that BRD4 controls RUNX2 by binding to the enhancers (ENHs) and each RUNX2 ENH is potentially controlled by a distinct set of TFs and c-JUN as the principal pivot of this regulatory platform. PMID: 28981843
- AP-1 likely plays a more important role in the AR cistrome in fibroblasts. PMID: 27634452
- Elevated levels of bile acid increase the tumorigenic potential of pancreatic cancer cells by inducing FXR/FAK/c-Jun axis to upregulate MUC4 expression. PMID: 27185392
- Immunohistochemistry was employed to analyze cFos, cJun, and CD147 expression in 41 UCB cases and 34 noncancerous human bladder tissues. PMID: 28358415
- Taken together, these findings indicate that LT reduces c-Jun protein levels via two distinct mechanisms, thereby inhibiting critical cell functions, including cellular proliferation. PMID: 28893904
- Expression of either dominant-negative or constitutively active mutants of Nrf2, ATF4, or c-Jun confirmed that distinct transcription units are regulated by these transcription factors. PMID: 27278863
- Mutually exclusive transcriptional regulation by AP-1 (cjun/cfos) and non-canonical NF-kappaB (RelB/p52) downstream of MEK-ERK and NIK-IKK-alpha-NF-kappaB2 (p100) phosphorylation, respectively, was responsible for persistent Ccl20 expression in the colonic cells. PMID: 27590109
- Glucocorticoid receptor (GR) is recruited to activator protein-1 (AP-1) target genes in a DNA-binding-dependent manner. PMID: 28591827
- These results suggested that hyperphosphatemia in the patients with CKD suppresses bone resorption by inhibiting osteoclastogenesis, and this impairs the regulation of bone metabolism. PMID: 28939042
- These results suggest that Bacteroides fragilis enterotoxin induced accumulation of autophagosomes in endothelial cells, but activation of a signaling pathway involving JNK, AP-1, and CHOP may interfere with complete autophagy. PMID: 28694294
- Overall, our results suggest that miR-4632 plays an important role in regulating HPASMC proliferation and apoptosis by suppression of cJUN, providing a novel therapeutic miRNA candidate for the treatment of pulmonary vascular remodeling diseases. It also implies that serum miR-4632 has the potential to serve as a circulating biomarker for PAH diagnosis. PMID: 28701355
- Findings suggest that AP-1 factors are regulators of RNA polymerase III (Pol III)-driven 5S rRNA and U6 snRNA expression with a potential role in cell proliferation. PMID: 28488757
- Our results indicate that assessing AP1 and PEA3 transcription factor status might be a good indicator of OAC status. However, we could not detect any associations with disease stage or patient treatment regime. This suggests that the PEA3-AP1 regulatory module more likely contributes more generally to the cancer phenotype. In keeping with this observation, depletion of ETV1 and/or ETV4 causes an OAC cell growth defect. PMID: 28859074
- shRNA-mediated inhibition of JUN decreases AML cell survival and propagation in vivo. These data uncover a previously unrecognized role of JUN as a regulator of the unfolded protein response. PMID: 27840425
- These findings demonstrate an essential role for the ERK pathway together with c-JUN and c-FOS in the differentiation activity of LukS-PV. PMID: 27102414
- The present study defines the minimal TIM-3 promoter region and demonstrates its interaction with c-Jun during TIM-3 transcription in CD4(+) T cells. PMID: 27243212
- Taken together, our data demonstrate that JNK regulates triple-negative breast cancer (TNBC) tumorigenesis by promoting CSC phenotype through Notch1 signaling via activation of c-Jun and indicate that JNK/c-Jun/Notch1 signaling is a potential therapeutic target for TNBC. PMID: 27941886
- Regulation of osteosarcoma cell lung metastasis by the c-Fos/AP-1 target FGFR1. PMID: 26387545
- c-jun promoted FOXK1-mediated proliferation and metastasis via orthotopic implantation. PMID: 27882939
- Data provide evidence that AP-1 is a key determinant of endocrine resistance of breast cancer cells by mediating a global shift in the estrogen receptor transcriptional program. PMID: 26965145
- Comparison of how AP-1 (Jun/Jun dimer) and Epstein-Barr virus Zta recognize methyl groups within their cognate response elements. PMID: 28158710
Q&A
What is c-Jun and why is phosphorylation at Y170 significant?
c-Jun is a proto-oncogene first identified as the cellular homolog of the avian sarcoma virus v-Jun oncogene. The c-Jun protein, along with c-Fos, forms the AP-1 transcriptional complex that binds to the TGACTCA consensus sequence (TRE - TPA response element) present in numerous gene promoters .
How does c-Jun phosphorylation at Y170 differ from other known phosphorylation sites?
c-Jun contains multiple phosphorylation sites that regulate its activity and stability through different mechanisms:
Unlike the well-established JNK-mediated phosphorylation at serines 63 and 73, which clearly regulates c-Jun stability and transcriptional activity, Y170 phosphorylation appears to have more nuanced effects. Experimental evidence using Y170F (unphosphorylable) and Y170D (phosphomimetic) mutations suggests that altering this residue does not significantly impact c-Jun's turnover or abolish its functions in regulating cellular proliferation and survival .
What are the optimal applications for Phospho-JUN (Y170) antibodies and their recommended dilutions?
Phospho-JUN (Y170) antibodies have been successfully employed in multiple research applications with specific optimization parameters:
For Western blotting, optimization may be required based on lysate concentration and specific experimental conditions. When performing IHC, appropriate antigen retrieval techniques should be employed to ensure optimal detection of phosphorylated epitopes, which can be more sensitive to fixation conditions than total protein .
How should Phospho-JUN (Y170) antibodies be stored and handled to maintain reactivity?
Proper storage and handling are critical for maintaining antibody performance:
-
Long-term storage: Store at -20°C or -80°C for up to one year from receipt date .
-
Short-term storage: For frequent use, store at 4°C for up to one month .
-
Avoid repeated freeze-thaw cycles as they can degrade antibody quality and performance .
-
Most commercial preparations contain 50% glycerol, 0.5% BSA, and 0.02% sodium azide in PBS to stabilize the antibody .
-
When aliquoting for long-term storage, use sterile tubes and maintain aseptic conditions .
Thaw aliquots completely before use and mix gently by inverting the tube rather than vortexing, which can denature the antibody protein structure .
What controls should be included when using Phospho-JUN (Y170) antibodies?
Rigorous experimental design requires appropriate controls:
-
Positive control: Lysates from cells treated with agents known to induce Y170 phosphorylation (e.g., certain growth factors or stress inducers). PC3, HEK293T, and kidney tissue lysates have been validated as positive controls .
-
Negative controls:
-
Specificity controls:
-
Cross-reactivity assessment: Though the antibody is reported to be specific for Y170 phosphorylation, testing on samples with known phosphorylation status at other c-Jun sites (e.g., Y26, S63/73) can confirm specificity .
How can researchers validate phospho-specificity in experimental systems?
Validating phospho-specificity requires multiple complementary approaches:
-
In vitro kinase assays: Incubate recombinant c-Jun with active CSK or c-Abl kinases in the presence of ATP, then probe with the antibody to confirm detection of newly phosphorylated Y170 .
-
Mutagenesis studies: Express wild-type c-Jun alongside Y170F (unphosphorylatable) and Y170D (phosphomimetic) mutants in cellular systems to confirm antibody specificity .
-
Phosphatase treatments: Treat positive control samples with lambda phosphatase prior to immunoblotting. Loss of signal confirms phospho-specificity .
-
Peptide competition: Pre-incubate the antibody with excess phosphorylated and non-phosphorylated peptides corresponding to the Y170 region. Specific signal should be blocked only by the phosphopeptide .
-
Signal correlation: Treatment conditions that modulate c-Jun activity should show corresponding changes in Y170 phosphorylation if the antibody is truly phospho-specific .
How should researchers interpret contradictory findings regarding Y170 phosphorylation and c-Jun stability?
The literature contains seemingly contradictory findings regarding Y170 phosphorylation:
-
Original hypothesis: Earlier biochemical studies suggested that CSK-mediated phosphorylation of c-Jun at Y170 promotes c-Jun degradation, with Y170 being identified as the major phosphorylation site .
-
Contradictory evidence: Subsequent research using Y170F (unphosphorylable) and Y170D (phosphomimetic) mutants in c-jun(-/-) embryonic fibroblasts concluded that "Y170 residue is not a critical determinant of c-Jun stability" .
When interpreting these contradictions, researchers should consider:
-
Different experimental systems (in vitro vs. cellular)
-
Cell type-specific effects
-
Temporal dynamics of phosphorylation
-
Interaction with other post-translational modifications
A comprehensive approach should examine the functional consequences of Y170 phosphorylation beyond protein stability, including potential effects on DNA binding, transcriptional activity, and protein-protein interactions .
What are the common technical challenges when detecting Y170 phosphorylation and how can they be overcome?
Several technical challenges may arise when detecting Y170 phosphorylation:
For particularly challenging samples, consider using a sequential immunoprecipitation approach: first immunoprecipitate with anti-c-Jun antibody, then probe with anti-phosphotyrosine antibody, or vice versa .
How does Y170 phosphorylation functionally interact with other c-Jun post-translational modifications?
c-Jun is subject to extensive post-translational regulation, with Y170 phosphorylation potentially interacting with other modifications:
Advanced research should employ phospho-proteomic approaches to comprehensively map all c-Jun modifications under various cellular conditions and investigate their functional interrelationships.
What emerging techniques can enhance detection and functional analysis of Y170-phosphorylated c-Jun?
Several cutting-edge techniques offer new opportunities for studying Y170 phosphorylation:
-
Proximity ligation assays (PLA): This technique can detect Y170-phosphorylated c-Jun with greater sensitivity than conventional immunodetection methods, allowing visualization of endogenous phosphorylation events in situ.
-
CRISPR-Cas9 genome editing: Generation of Y170F knock-in cell lines provides a clean genetic system to study the functional significance of this phosphorylation site without overexpression artifacts.
-
Phospho-specific intrabodies: These genetically encoded sensors can be used to track Y170 phosphorylation in living cells with temporal and spatial resolution.
-
Mass spectrometry-based phosphoproteomics: This approach can quantitatively assess Y170 phosphorylation alongside other modifications, providing a holistic view of c-Jun's modification state.
-
Single-molecule imaging: Techniques like single-molecule fluorescence resonance energy transfer (smFRET) can probe how Y170 phosphorylation affects c-Jun conformation and interactions with DNA and other proteins.
These advanced techniques can help resolve the functional significance of Y170 phosphorylation in different cellular contexts and in response to various stimuli.
