Phospho-FOS (Thr232) Antibody
Description
Antibody Overview
Phospho-FOS (Thr232) Antibody is a rabbit polyclonal antibody designed to selectively recognize the phosphorylated form of c-Fos at Thr232 . This modification occurs in response to Ras-activated pathways and plays a regulatory role in AP-1 transcriptional activity . The antibody is validated for use in Western blot (WB), immunohistochemistry (IHC), immunofluorescence (IF), ELISA, and chromatin immunoprecipitation (ChIP) assays .
Mechanism of Action
-
Phosphorylation at Thr232 by MAPK/RSK kinases stabilizes c-Fos, enhancing its transcriptional activity by antagonizing sumoylation .
-
This modification is critical for AP-1 complex formation with Jun proteins, enabling DNA binding at AP-1/SMAD promoter sites to regulate TGF-β signaling .
Functional Roles
-
Cell Proliferation: Drives phospholipid synthesis via activation of CDS1 and PI4K2A at the endoplasmic reticulum .
-
Signal Transduction: Integrates Ras/MAPK pathways with TGF-β-mediated gene expression .
Experimental Validation
-
Western Blot: Detects phosphorylated c-Fos in EGF-stimulated SKBR3 cells, with specificity confirmed via peptide competition assays .
-
Immunofluorescence: Localizes phospho-c-Fos to nuclei in EGF-treated HeLa cells .
-
ChIP Analysis: Identifies phospho-c-Fos binding to IL-6 and MMP9 promoters in A431 cells .
Key Findings
Product Specs
Target Background
- Research findings suggest a human bone tumor characterized by mutations of FOS and FOSB. PMID: 29858576
- gammadelta T cells suppress iDCs osteoclastogenesis by downregulating the RANK/cFos/ATP6V0D2 signaling pathway. PMID: 30066839
- 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
- Low c-fos expression is associated with Oral Squamous Cell Carcinoma. PMID: 29582647
- A study demonstrated that c-Fos was highly expressed in most ovarian epithelial carcinoma cases and was significantly correlated with Lewis y. Furthermore, the results indicated that c-Fos interacted with the FUT1 promoter. Silencing of c-Fos prevented TGF-beta1-induced Lewis y expression. PMID: 29130097
- These findings indicate that the c-Fos/miR-22/MDC1 axis plays a relevant role in DNA repair in terminally differentiated cells, potentially contributing to our understanding of the molecular mechanisms underlying the downregulation of DNA repair in differentiated cells. PMID: 28637007
- Our results strongly suggest a novel role of c-Fos as a regulator of epithelial-mesenchymal transition and cancer stem cell (CSC) reprogramming in Head and neck squamous cell carcinoma (HNSCC) cells, which may hold potential as a CSC-directed therapeutic approach to improve HNSCC treatment. PMID: 27965308
- High c-fos expression is associated with malignant glioma. PMID: 27602752
- Immunohistochemistry was employed to analyze cFos, cJun, and CD147 expression in 41 UCB cases and 34 noncancerous human bladder tissues. PMID: 28358415
- Data suggest that knockdown of c-Fos inhibited cell proliferation, migration, and invasion, and promoted apoptosis of OS cells accompanied by altered expression of Wnt2 and Fzd9. PMID: 28665975
- 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
- A novel function of KDM2B in the negative regulation of cell proliferation by assembling an E3 ligase to target c-Fos protein degradation that is antagonized by mitogenic stimulations. PMID: 26725323
- NF-Y Binding Site Architecture Defines a C-Fos Targeted Promoter Class. PMID: 27517874
- c-fos underexpression is associated with Myelodysplastic Syndrome. PMID: 27513856
- miR-101 is downregulated in bladder cancer cells and has an inhibitory role in the regulation of bladder cancer cell proliferation and invasion via directly targeting c-FOS. PMID: 27485165
- We observed that c-jun or c-fos was significantly associated with lymph node metastasis, and coexpression of c-jun/c-fos, or c-jun/c-fos/p53 were significantly associated with lymph node metastasis, poor differentiation, and clinical stage. PMID: 27558649
- CRAC channel blockade also suppressed Oxo-M-induced c-fos and interleukin-2 expression. PMID: 27474128
- The results indicate that 17beta-estradiol-induced endometrial stromal cell invasion is dependent on c-fos-mediated MMP-9 expression. PMID: 26917263
- FOS is a downstream effector of high glucose stimulation in peritoneal mesothelial cells that contributes to TGF-beta1 production. PMID: 26018137
- VEGF-induced endothelial migration is mediated primarily by induction of JunB whereas the promotion of endothelial proliferation by VEGF is mediated by JunB-independent AP-1 family members. PMID: 26860974
- c-Fos can protect against HDAC3 neurotoxicity. PMID: 25592718
- These results indicate that IL-17A enhances COX2 expression and PGE2 production via the p38/c-Fos and JNK/c-Jun signaling pathways in NP cells to mediate intervertebral disc inflammation. PMID: 26988982
- The results of this study suggest that FOS is among the candidate genes of schizophrenia and that changes in the expression of c-Fos protein may contribute to molecular mechanisms of schizophrenia-related alterations in synaptic plasticity. PMID: 25706621
- Increased c-Fos expression is through TRPM3-mediated stimulation of the c-Fos promoter. PMID: 26493679
- A novel AP-1 binding site at -1363 bp of the human TF promoter region was identified. PMID: 26631725
- Simultaneous high expression of ID1 and c-Jun or c-Fos was correlated with poor survival in esophageal squamous cell carcinoma patients. PMID: 26858249
- miR-146a has a role in targeting Fos expression in human cardiac cells. PMID: 26112171
- The translocation causes truncation of the FOS protein, with loss of the transactivation domain, which is thereby a novel mechanism involved in tumorigenesis. PMID: 26173738
- ERK1 and ERK2 regulated the expression of c-Fos and c-Jun proteins in human cervical cancer cells. PMID: 25647783
- O-GlcNAcylation of MLL5beta at T440 residue is critical for MLL5 recruitment to the HPV16/18-long control region through its interaction with AP-1. PMID: 25670814
- The RNA binding complexes NF45-NF90 and NF45-NF110 associate dynamically with the c-fos gene and function as transcriptional coactivators. PMID: 26381409
- Data show that interleukin-1 receptor type 2 (IL1R2) forms a complex with c-Fos proto-oncogene protein and activates the interleukin-6 (IL-6) and vascular endothelial growth factor A (VEGF-A) promoters. PMID: 26209639
- Data indicate that deregulation of transcription factor AP-1 and microRNA-21-mediated axis led to an enhanced cell growth in hepatocellular carcinoma (HCC). PMID: 25544773
- These results establish c-Fos homodimers as a novel form of the AP-1 complex that may be an autonomous transcription factor in c-Fos-overexpressing tissues and could contribute to tumor development. PMID: 26303532
- Endoplasmic reticulum stress activates the hepatic AP-1 complex via MAPK-dependent signaling pathways. PMID: 25077945
- Co-expression of c-Fos or Fra1 was able to cooperate with TAp73 in potentiating cellular growth, similarly to c-Jun. These data together suggest that TAp73 plays a vital role in activation of AP-1 target genes via direct binding to c-Jun. PMID: 26018080
- The light-induced FOS response in melanopsin expressing HEK-293 cells is correlated with melanopsin quantity and dependent on light duration and irradiance. PMID: 24909488
- c-Fos promotes the progression of viral transcription from early to late stages and accelerates viral lytic replication upon sustained ORF45-ERK-RSK activation during the Kaposi's Sarcoma-Associated Herpesvirus lytic life cycle. PMID: 25903346
- By targeting the proto-oncogene Fos, miR-101 is involved in G1-to-S phase transition in cervical cancer cells in vitro. PMID: 24987920
- Data suggest that p38 MAP kinase regulates c-Fos/cellular oncogene fos mRNA stability/decay by affecting the state of phosphorylation of ELAVL1/HuR (Hu antigen R). PMID: 25588078
- CDK12 plays an important role in cotranscriptional processing of c-FOS transcripts. PMID: 25384976
- We found significant negative correlations regarding the expression of the genes COMT, MAOB, DRD4, DRD5, and FOS, indicating that increased schizotypy coincides with higher levels of dopaminergic dysregulation at the mRNA level. PMID: 24630741
- Results support the proposal that cooperative signaling of both NF-kappaB and AP1 (via p38alpha) amplifies STIM1 expression in ECs and, thereby, contributes to the lung vascular hyperpermeability response during sepsis. PMID: 25016017
- SMAR1 has a role in repressing c-Fos-mediated HPV18 E6 transcription through alteration of chromatin histone deacetylation. PMID: 25157104
- This study indicates that increased expression of c-Fos, p-c-Jun, members of the AP-1 transcriptional factor, and p-JNK is associated with neuronal degeneration in the ganglion cell layer of retinas in diabetic patients. PMID: 24073601
- S100A4, FOS, and CXCR4, playing a major role in tumor progression and metastasis, are downregulated by sorafenib. PMID: 24378831
- The IL-1beta/p38/AP-1(c-fos)/MMP2 & MMP9 pathway plays an important role in metastasis in gastric adenocarcinoma. PMID: 24479681
- The distinct requirement of NF-kappaB for mouse and human c-fos regulation. PMID: 24386331
- c-Fos, a well-known AP-1 transcription factor, has emerged as a unique protein with the capacity to associate with specific enzymes of the phospholipid synthesis pathway at the endoplasmic reticulum and activate their synthesis. (Review) PMID: 24886961
- Inflammation mediators act through c-Fos to increase VEGF production in peritoneal mesothelium. PMID: 23760290
Q&A
What is Phospho-FOS (Thr232) Antibody and what does it detect?
Phospho-FOS (Thr232) Antibody is a rabbit polyclonal antibody specifically designed to detect endogenous levels of the FOS protein only when phosphorylated at threonine 232 . This site-specific phosphorylation recognition is achieved through specialized production methods where antibodies are generated by immunizing rabbits with synthetic phosphopeptides conjugated to KLH (Keyhole Limpet Hemocyanin) . The antibody undergoes rigorous purification using affinity chromatography with epitope-specific phosphopeptides, with non-phospho specific antibodies being removed through non-phosphopeptide chromatography . This ensures high specificity for the phosphorylated form of FOS at threonine 232.
The target protein, FOS (also known as c-Fos), functions as a nuclear phosphoprotein that forms a tight but non-covalently linked complex with the JUN/AP-1 transcription factor . This antibody enables researchers to specifically monitor the phosphorylation state at Thr232, which is a critical regulatory site affecting FOS transcriptional activity.
What are the common applications for Phospho-FOS (Thr232) Antibody?
Phospho-FOS (Thr232) Antibody is validated for multiple experimental applications, primarily:
-
Western Blot (WB): Useful for detecting phosphorylated FOS in cell and tissue lysates with recommended dilution ranges of 1:500-1:3000
-
Immunohistochemistry (IHC): Enables visualization of phospho-FOS in tissue sections with recommended dilution ranges of 1:50-1:100
-
ELISA: Allows quantitative detection with dilution ranges up to 1:20000
The antibody has been successfully tested on various cell lines including Jurkat and COS7 cells, demonstrating its utility across different experimental systems . For immunohistochemical applications, it has been validated on formalin-fixed and paraffin-embedded human breast carcinoma tissue . These diverse applications make this antibody a versatile tool for researchers studying signaling pathways involving FOS phosphorylation.
What species reactivity does Phospho-FOS (Thr232) Antibody exhibit?
Phospho-FOS (Thr232) Antibody demonstrates cross-reactivity with multiple species, making it valuable for comparative studies. The antibody has confirmed reactivity with:
This multi-species reactivity is particularly advantageous for researchers conducting translational studies or using animal models to investigate conserved signaling pathways involving FOS phosphorylation. When designing experiments with different species, it is recommended to validate antibody performance in each specific model system, as slight variations in epitope recognition may occur despite the high conservation of this phosphorylation site across species.
What is the biological significance of FOS and its phosphorylation at Thr232?
FOS is a key component of the Activator Protein-1 (AP-1) transcription factor complex, which regulates gene expression related to cellular proliferation, differentiation, and transformation . FOS heterodimerizes with JUN to form AP-1, and in this heterodimer, both proteins' basic regions interact with symmetrical DNA half-sites .
Phosphorylation at Thr232 has distinct functional consequences:
-
It antagonizes sumoylation, another post-translational modification affecting FOS function
-
It is regulated by ERK MAPK, which affects the nuclear localization of c-FOS and consequently its transcriptional activity
FOS also participates in TGF-beta signaling by forming a multimeric complex with SMAD3/SMAD4/JUN at the AP1/SMAD-binding site . Additionally, it has a critical role in regulating the development of cells destined to form and maintain the skeleton . In growing cells, FOS activates phospholipid synthesis, possibly by activating CDS1 and PI4K2A, an activity requiring tyrosine dephosphorylation and association with the endoplasmic reticulum .
How does phosphorylation at Thr232 interact with other post-translational modifications of FOS?
Phosphorylation at Thr232 represents just one of multiple regulatory modifications affecting FOS function. This site interacts with a complex network of other phosphorylation events and post-translational modifications:
FOS undergoes phosphorylation at multiple sites, including Ser-362 and Ser-374 by MAPK1/2 and RSK1/2, which leads to protein stabilization . Phosphorylation at Ser-374 appears to be the major site for protein stabilization upon NGF stimulation . Interestingly, phosphorylation at Ser-362 and Ser-374 primes further phosphorylations on Thr-325 and Thr-331 by promoting docking of MAPK to the DEF domain .
The phosphorylation at Thr232 specifically interacts with sumoylation processes, with Thr232 phosphorylation antagonizing sumoylation . This indicates a regulatory mechanism where different post-translational modifications counterbalance each other to fine-tune FOS activity. Additionally, in osteoblasts, phosphorylation on Ser-362 by RSK2 contributes to osteoblast transformation .
Researchers investigating these complex interactions should consider employing multiple phospho-specific antibodies to fully characterize the phosphorylation status of FOS in their experimental systems.
What experimental controls should be included when using Phospho-FOS (Thr232) Antibody?
To ensure rigorous and reproducible results with Phospho-FOS (Thr232) Antibody, researchers should implement several critical controls:
-
Phosphatase Treatment Control: Treating a portion of your sample with lambda phosphatase before immunoblotting should eliminate the signal if the antibody is truly phospho-specific.
-
Blocking Peptide Validation: Using a synthetic blocking peptide like the Human c-Fos (phospho T232) peptide in blocking experiments can confirm specificity . This approach involves pre-incubating the antibody with the phosphopeptide before application to the sample, which should neutralize the antibody and diminish the signal.
-
Positive Controls: Include samples known to have high levels of phosphorylated FOS at Thr232, such as cells treated with growth factors or stimuli that activate the MAPK pathway . Jurkat and COS7 cell extracts have been validated as suitable positive controls .
-
Negative Controls: Include samples with low or absent phosphorylation at this site, such as serum-starved cells or cells treated with specific ERK MAPK inhibitors .
-
Loading Controls: Include antibodies against total FOS and housekeeping proteins like beta-actin or GAPDH to normalize for protein loading and to distinguish between changes in phosphorylation versus changes in total protein expression.
What are the optimal protocols for Western Blot using Phospho-FOS (Thr232) Antibody?
For optimal Western Blot results with Phospho-FOS (Thr232) Antibody, the following protocol recommendations should be considered:
Sample Preparation:
-
Lyse cells in a buffer containing phosphatase inhibitors to preserve phosphorylation status
-
For best results, stimulate cells with appropriate agonists known to induce FOS phosphorylation (growth factors, serum, etc.)
-
Expected molecular weight of FOS is approximately 48kD on SDS-PAGE
Protocol Parameters:
-
Use 4-20% gradient gels for optimal resolution
-
Transfer proteins to PVDF membrane for better protein retention
-
Block with 5% BSA in TBST rather than milk, as milk contains phosphatases that could reduce signal
-
Incubate with primary antibody overnight at 4°C for optimal binding
-
Use HRP-conjugated anti-rabbit secondary antibodies
Visualization:
-
For weak signals, consider using enhanced chemiluminescence (ECL) substrates with higher sensitivity
-
When quantifying results, ensure exposure times are within the linear range of detection
Troubleshooting:
-
If background is high, increase blocking time and wash duration
-
If signal is weak, reduce antibody dilution or increase protein loading
-
Consider using signal enhancement systems for low abundance phosphoproteins
How can Phospho-FOS (Thr232) Antibody be used in multiplexed signaling pathway analysis?
Phospho-FOS (Thr232) Antibody can be effectively integrated into multiplexed approaches for comprehensive signaling pathway analysis:
The T-Cell Receptor Phospho Antibody Array represents one such platform, featuring 213 site-specific and phospho-specific antibodies including Fos(Thr232) . This high-throughput ELISA-based antibody array allows for qualitative protein expression profiling across multiple signaling pathways simultaneously. With six replicates per antibody and fluorescent detection, this approach provides robust data for comparing protein expressions between control and treated samples .
For researchers focusing on specific signaling cascades, Phospho-FOS (Thr232) Antibody can be used alongside antibodies targeting:
-
Upstream regulators: ERK MAPK (Thr202/Tyr204), RSK, and RAS pathway components
-
Parallel pathways: JNK1/2/3 (Thr183/Tyr185) and p38 MAPK to assess cross-talk
-
Other AP-1 components: c-Jun phosphorylation sites (Ser63, Ser73, Thr91, Thr93, etc.)
-
Downstream effectors: Target genes regulated by AP-1
These multiplexed approaches enable researchers to build a comprehensive picture of signaling dynamics in various experimental conditions, providing insights into the temporal and spatial regulation of FOS phosphorylation in the context of broader cellular signaling networks.
What are the considerations for using Phospho-FOS (Thr232) Antibody in different tissue types?
When applying Phospho-FOS (Thr232) Antibody to different tissue types, researchers should consider several tissue-specific factors:
Tissue Fixation and Processing:
-
For immunohistochemistry, the antibody has been validated on formalin-fixed, paraffin-embedded human breast carcinoma tissue
-
Different fixation protocols may affect epitope accessibility; antigen retrieval methods should be optimized for each tissue type
-
Fresh frozen tissues may provide better preservation of phospho-epitopes compared to fixed tissues
Tissue-Specific Expression Patterns:
-
FOS expression and phosphorylation levels vary considerably across different tissues and cell types
-
Skeletal tissues may be of particular interest given FOS's critical function in regulating the development of cells destined to form and maintain the skeleton
-
Brain tissues often exhibit high basal and inducible FOS expression in specific neuronal populations
Background Considerations:
-
Some tissues (like liver) have high endogenous peroxidase activity that could create background in IHC; appropriate blocking steps are essential
-
Tissues with high levels of endogenous biotin (like kidney and liver) may require biotin blocking steps if using biotin-based detection systems
Detection Sensitivity:
