ier2 Antibody

What is IER2 and why are antibodies against it important for research?

IER2 is a rapidly but transiently induced protein in response to various growth factors and mitogenic stimuli. The protein contains multiple PEST sequences (making it short-lived), a putative bipartite nuclear localization signal (NLS), and has limited homology to JunB and JunD. Despite lacking obvious DNA-binding motifs, IER2 has been implicated in transcriptional regulation and protein phosphatase PP2A activity modulation .

What are the key structural features of IER2 that antibodies typically target?

IER2 proteins contain several distinctive structural features that antibodies can target:

• Nuclear localization signal (NLS): A bipartite sequence critical for the protein's nuclear translocation

• PEST sequences: Multiple degradation-promoting regions that contribute to its short half-life

• Conserved domains: Regions with homology to transcription factors like JunB and JunD

In medaka fish, the deduced IER2 amino acid sequence consists of 171 residues with highly conserved regions, suggesting evolutionary importance across species . When designing or selecting IER2 antibodies, researchers should consider which domain is most relevant to their research question, as nuclear localization of IER2 is particularly critical for both senescence induction and osteopontin secretion in melanoma cells .

How does IER2 expression vary across cell types and experimental conditions?

IER2 expression patterns show significant variability:

IER2 expression is typically low in normal cells but can be rapidly induced by various stimuli, returning to baseline within 48 hours. In experimental settings, researchers often use daily treatment with inducers (like RheoSwitch Ligand) to maintain sustained expression . Analysis using the Heuristic Online Phenotype Prediction algorithm revealed significantly higher IER2 expression in melanoma cells with intermediate or invasive phenotypes compared to those with primarily proliferative characteristics .

How can IER2 antibodies be used to study its role in melanoma progression?

IER2 antibodies serve as essential tools for investigating several aspects of melanoma progression:

What methodologies can detect the interaction between IER2 and the p53 pathway?

Researchers can employ several approaches to investigate IER2-p53 interactions:

• Co-immunoprecipitation: Using IER2 antibodies to pull down protein complexes followed by p53 detection can reveal direct or indirect interactions.

• Western blot analysis: IER2 expression in B16 melanoma cells leads to p53 stabilization and activation. Sequential western blotting with antibodies against IER2, total p53, phospho-p53 (Ser15, Ser392), MDM2 isoforms, and p21 can map the complete pathway activation .

• Pharmacological inhibition: Combining IER2 antibody detection with p53 inhibitors or activators helps establish causal relationships. Studies show that p53-deficient cells (p53 null) remained unaffected by IER2 overexpression, while re-introduction of wild-type p53 enhanced IER2's ability to induce senescence .

• Chemotherapy response: IER2 promotes earlier stabilization and activation of p53 in response to doxorubicin and camptothecin, demonstrating its role in p53 regulation under stress conditions. Antibodies against IER2 and p53 can track this temporal relationship .

These methodologies collectively revealed that wild-type p53 is required for efficient IER2-induced senescence, providing critical insights into the molecular mechanisms of IER2 in cancer progression.

How do IER2 antibodies help investigate the senescence-associated secretory phenotype (SASP)?

IER2 antibodies are instrumental in studying the complex relationship between IER2, senescence, and SASP:

• Identification of senescent populations: IER2 antibodies combined with senescence markers can identify the subset of cells undergoing senescence following IER2 expression. In melanoma models, sustained IER2 expression induced senescence in a fraction of cells, which then developed a characteristic secretory profile .

• Secretome analysis: After identifying IER2-expressing senescent cells, researchers can analyze their secretome. Gene set enrichment analysis revealed that IER2-induced genes involve processes including lipid metabolism, immune/inflammatory responses, and cytokine production—characteristic of SASP .

• Osteopontin measurement: A major component of the IER2-driven SASP is osteopontin, which plays a central role in driving melanoma cell invasion. Antibodies against both IER2 and osteopontin can correlate expression levels with invasive capacity .

• Paracrine effect quantification: IER2 antibodies help identify which cells (senescent vs. non-senescent) are responsible for promoting invasion. Studies showed that osteopontin secreted by IER2-expressing senescent cells strongly stimulates the migration and invasion of non-senescent melanoma cells .

This research revealed the mechanism by which IER2 contributes to melanoma progression—not by making all cells more invasive, but by inducing senescence in some cells that then stimulate invasion in others through secreted factors.

What are the optimal protocols for IER2 immunodetection in different sample types?

Effective IER2 detection requires optimized protocols for different experimental scenarios:

When detecting IER2 in cellular contexts, researchers should consider that cell density influences subcellular localization—cells released from contact inhibition show translocation of IER2 from cytoplasm to nucleus . For experimental models with inducible IER2 expression, maximum expression typically occurs 6 hours after induction and diminishes to basal levels within 48 hours, necessitating daily induction for sustained expression studies .

How can researchers validate IER2 antibody specificity for experimental applications?

Thorough validation of IER2 antibodies is critical due to its low basal expression and transient induction pattern:

• Positive controls: Use cells with verified IER2 overexpression (e.g., RheoSwitch Ligand-inducible systems) to confirm antibody reactivity under controlled conditions .

• Negative controls: Employ p53-deficient cell lines like 67NR mouse breast adenocarcinoma cells, which show minimal response to IER2 induction, as specificity controls .

• Subcellular localization: Validate that the antibody can detect the known translocation pattern of IER2 from cytoplasm to nucleus upon release from contact inhibition .

• Correlation with mRNA levels: Compare protein detection with RT-qPCR quantification of IER2 mRNA following induction.

• Blocking peptides: Use synthetic peptides corresponding to the antibody epitope to confirm specificity in western blot and immunohistochemistry applications.

• Cross-species reactivity: Consider that IER2 contains highly conserved sequences across species, as demonstrated in the medaka fish study, which may affect antibody specificity when working with different model organisms .

What experimental designs best capture IER2's dynamic expression and localization?

IER2's transient expression and variable localization require specialized experimental designs:

• Time-course studies: Since IER2 expression peaks at approximately 6 hours after induction and returns to baseline within 48 hours, time-course experiments with multiple sampling points are essential .

• Subcellular fractionation: Nuclear/cytoplasmic fractionation followed by western blotting can quantify IER2 translocation between cellular compartments.

• Live-cell imaging: For real-time tracking of IER2 dynamics, fluorescent protein fusions or antibody fragments can be employed, though care must be taken that tags don't interfere with the protein's NLS.

• Cell density manipulation: Experimental designs should control for and leverage the fact that cell density influences IER2 localization .

• Sustained expression models: For studying long-term effects of IER2, such as senescence induction, daily treatment with inducers like RSL is necessary to maintain expression beyond 24 hours .

• Co-detection systems: Simultaneous detection of IER2 and its downstream effects (p53 activation, osteopontin secretion) provides comprehensive mechanistic insights.

How does IER2 expression correlate with genetic alterations in melanoma?

Analysis of the TCGA skin cutaneous melanoma dataset revealed important correlations between IER2 expression and genetic alterations:

High IER2 expression is predominantly associated with wild-type BRAF and mutated NRAS status, consistent with the mutual exclusivity of BRAF and NRAS mutations in melanoma . Importantly, high IER2 expression was almost exclusively associated with wild-type p53 status, aligning with findings that p53 levels increase in melanoma cells upon sustained IER2 expression and that p53 is required for IER2-induced senescence .

This genetic context provides critical information for researchers using IER2 antibodies in patient sample characterization, suggesting that IER2 expression should be evaluated alongside these key genetic alterations to properly interpret its significance.

What cell signaling pathways interact with IER2 functions?

IER2 interacts with multiple signaling pathways critical to melanoma progression:

• MAPK pathway: IER2-induced senescence involves activation of the MAPK pathway. Treatment with MEK inhibitors significantly reduces IER2-induced senescence in melanoma cells, demonstrating a mechanistic link .

• AKT pathway: The AKT pathway also plays a role in IER2-induced senescence. AKT inhibitors reduce the senescence phenotype induced by IER2 expression .

• p53 signaling: IER2 expression increases TRP53 (mouse homolog of p53) levels while decreasing expression of MDM2, a negative regulator of p53. This leads to p53 stabilization and activation, evidenced by phosphorylation at key sites .

• Integrin-FAK-Src pathway: In hepatocellular carcinoma, IER2 regulates migration and invasion via an integrin β1-focal adhesion kinase (FAK)-Src-paxillin signaling pathway and Rho GTPases .

When both MAPK and AKT pathways are inhibited simultaneously, there is a significant decrease in polyploidy and senescence-associated β-galactosidase activity in IER2-expressing cells, indicating these pathways are synergistically required for IER2-induced senescence .

What role does IER2 play in the response to chemotherapeutic agents?

IER2 significantly influences cellular responses to chemotherapeutic agents through p53-dependent mechanisms:

• Enhanced p53 activation: Elevated IER2 levels promote earlier stabilization and activation of p53 in response to chemotherapeutics like doxorubicin and camptothecin .

• Accelerated cell cycle regulator induction: IER2 expression results in an earlier increase in p21 levels following chemotherapy treatment, potentially affecting cell cycle arrest timing .

• Stress response modulation: IER2 contributes to p53 regulation under both normal and stress conditions, suggesting a role in determining cellular fate following genotoxic insult .

These findings have important implications for cancer treatment, as IER2 expression levels may influence chemotherapy responsiveness. Researchers can use IER2 antibodies to assess whether IER2 expression levels could serve as predictive biomarkers for treatment response or whether targeting IER2 might enhance chemosensitivity in certain contexts.

What are emerging applications of IER2 antibodies in cancer research?

Several promising research directions are emerging for IER2 antibodies:

These applications could transform IER2 from a poorly investigated protein into a clinically relevant biomarker and potential therapeutic target.

How can researchers address current technical challenges in IER2 antibody development?

Several technical challenges must be overcome to advance IER2 antibody research:

• Low basal expression: IER2 typically has very low basal expression levels, making detection challenging. Development of high-affinity antibodies with improved sensitivity is needed.

• Short protein half-life: IER2 protein is short-lived due to multiple PEST sequences , complicating consistent detection. Optimized sample preservation and processing protocols are essential.

• Isoform specificity: Research into potential IER2 isoforms or post-translational modifications would improve antibody specificity.

• Cross-species applications: Given IER2's evolutionary conservation from fish to mammals , developing antibodies with controlled cross-species reactivity would facilitate comparative studies across model organisms.

• Nuclear localization-specific detection: Since nuclear localization is functionally critical , developing antibodies that specifically recognize the nuclear-translocated form could provide functional insights.

Addressing these challenges would significantly advance our understanding of IER2 biology and its role in cancer progression.