EIF2AK4 Antibody

What is EIF2AK4 and what is its primary function?

EIF2AK4 (eukaryotic translation initiation factor 2 alpha kinase 4) is a member of a family of kinases that phosphorylate the alpha subunit of eukaryotic translation initiation factor-2 (EIF2), resulting in the downregulation of protein synthesis. The protein primarily responds to amino acid deprivation by binding uncharged transfer RNAs, but can also be activated by glucose deprivation and viral infection. With a calculated molecular weight of 187 kDa, this protein (also known as GCN2) plays a crucial role in cellular stress responses through the integrated stress response (ISR) pathway . Its genetic location is on chromosome 15q15.1, containing 39 exons in total .

What are the key disease associations of EIF2AK4?

EIF2AK4 mutations have significant clinical implications, particularly in pulmonary diseases. Biallelic mutations of the gene are causal in two ultra-rare subtypes of pulmonary arterial hypertension (PAH): pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis . Additionally, EIF2AK4 variants of unknown significance have been identified in patients with classical PAH, though their pathogenic significance remains under investigation . The gene has also been associated with susceptibility to Ewing sarcoma, corneal curvature variations in Australians, body mass index variations in Chinese populations, and responses to haloperidol treatment .

How should EIF2AK4 antibodies be stored and handled for optimal performance?

For optimal performance, EIF2AK4 antibodies such as the polyclonal 11174-1-AP should be stored at -20°C, where they remain stable for one year after shipment. The storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For these specific antibody preparations, aliquoting is unnecessary for -20°C storage, which simplifies handling protocols. Smaller size preparations (20μl) may contain 0.1% BSA as a stabilizer . Always minimize freeze-thaw cycles and keep the antibody on ice during experimental procedures to maintain binding capacity.

What validation methods are recommended to confirm EIF2AK4 antibody specificity?

To validate EIF2AK4 antibody specificity, researchers should implement a multi-faceted approach:

• Knockout/knockdown validation: Compare staining patterns between wild-type cells and EIF2AK4 knockout or siRNA knockdown cells. This approach has been effectively demonstrated in studies using eif2ak4-/- MEFs to confirm antibody specificity .

• Western blot analysis: Verify a single band of appropriate molecular weight (approximately 187 kDa) in tissues known to express EIF2AK4.

• Immunoprecipitation followed by mass spectrometry: To confirm the antibody is pulling down the correct protein.

• Peptide competition assay: Pre-incubate the antibody with immunizing peptide to block specific binding.

• Cross-validation with multiple antibodies: Use antibodies targeting different epitopes of EIF2AK4 to confirm consistent results.

For ChIP applications, additional controls as demonstrated in studies examining ATF4 binding to autophagy gene promoters should be included .

How can EIF2AK4 antibodies be utilized to study the integrated stress response (ISR) pathway?

EIF2AK4 antibodies are invaluable tools for investigating the integrated stress response pathway through several methodological approaches:

• Phosphorylation assays: Use phospho-specific antibodies to detect EIF2AK4 activation and subsequent EIF2A phosphorylation under various stress conditions. This approach has been successfully employed to monitor activation of the EIF2AK4-EIF2A-ATF4 pathway during bacterial infections .

• Immunoprecipitation: Isolate EIF2AK4 protein complexes to identify interacting partners during stress conditions.

• Chromatin immunoprecipitation (ChIP): As demonstrated in recent research, ChIP assays using anti-ATF4 antibodies can determine downstream transcriptional targets of the EIF2AK4-EIF2A-ATF4 pathway. Studies have shown that upon AIEC infection, ATF4 binds to promoters of autophagy genes including Sqstm1, Map1lc3b, Becn1, Atg3, and Atg7 .

• Immunofluorescence microscopy: Track EIF2AK4 subcellular localization during stress responses.

• Time-course experiments: Monitor EIF2AK4 activation, EIF2A phosphorylation, and ATF4 protein levels at different time points after stress induction .

These methodologies collectively provide a comprehensive view of EIF2AK4's role in mediating cellular stress responses through the ISR pathway.

What are the recommended protocols for using EIF2AK4 antibodies in studying autophagy mechanisms?

For studying autophagy mechanisms using EIF2AK4 antibodies, the following protocol framework is recommended based on recent research findings:

• Monitoring autophagy induction:

  • Use Western blotting to track LC3-II levels and SQSTM1 protein degradation with appropriate EIF2AK4 antibodies

  • Compare results between wild-type and EIF2AK4-depleted cells to establish EIF2AK4 dependency

• Autophagosome visualization:

  • Transfect cells with pEGFP-LC3 plasmid and monitor puncta formation under fluorescence microscopy

  • Quantify the number of puncta per cell following various stressors

  • Include appropriate controls such as rapamycin treatment (a known autophagy inducer)

• Gene expression analysis:

  • Perform RT-qPCR to measure expression levels of autophagy-related genes

  • Use ChIP assays with anti-ATF4 antibodies to assess binding to autophagy gene promoters

  • Include appropriate controls such as negative control IgG to confirm specificity

Research has demonstrated that EIF2AK4 is essential for inducing transcription of autophagy genes via ATF4 binding, thereby activating autophagy in response to stressors such as bacterial infection .

What are common challenges in EIF2AK4 detection and how can they be addressed?

When working with EIF2AK4 antibodies in cell lines with EIF2AK4 mutations or variants, it's important to verify that the antibody's epitope is not affected by the variant. This is particularly relevant given the emerging research on EIF2AK4 missense variants in pulmonary arterial hypertension .

How can researchers validate functional consequences of EIF2AK4 variants using antibody-based approaches?

Recent research has established a robust methodology for validating EIF2AK4 variants using antibody-based approaches:

• Expression level analysis: Use Western blotting with anti-EIF2AK4 antibodies to compare expression levels of wild-type and variant proteins to identify destabilized/misfolded variants .

• Phosphorylation status assessment: Utilize phospho-specific antibodies to detect EIF2A phosphorylation levels as a direct measure of EIF2AK4 kinase activity following stress induction.

• Downstream effector monitoring: Track ATF4 protein levels and target gene expression using appropriate antibodies and RT-qPCR to evaluate pathway functionality .

• Functional assay design: Create cellular stress conditions (amino acid deprivation, glucose deprivation) to activate EIF2AK4 and monitor downstream responses in cells expressing variant proteins compared to wild-type controls.

• Pharmacological intervention: Test variant responsiveness to EIF2AK4 modulators, as recent research has shown that some hypomorphic EIF2AK4 variants are amenable to paradoxical activation by type-1.5 GCN2 kinase inhibitors .

This experimental framework has demonstrated that EIF2AK4 variants can be functionally subclassified into three groups: misfolded, kinase-dead, and hypomorphic, which has important implications for disease understanding and potential therapeutic approaches .

How are EIF2AK4 antibodies being utilized in the study of pulmonary arterial hypertension (PAH)?

EIF2AK4 antibodies are increasingly central to advancing our understanding of pulmonary arterial hypertension, particularly in relation to genetic variants. Recent studies have employed these antibodies to:

• Characterize functional consequences of EIF2AK4 variants: Researchers have used antibody-based approaches to classify EIF2AK4 variants found in PAH patients into functional categories (likely benign, destabilized/misfolded, or kinase impaired), providing critical information for patient diagnosis and genetic counseling .

• Identify therapeutic targets: The discovery that some hypomorphic EIF2AK4 variants can be paradoxically activated by type-1.5 GCN2 kinase inhibitors has opened new avenues for potential targeted therapies .

• Develop improved diagnostic tools: Antibody-based functional assays now outperform computational prediction methods in determining the pathogenicity of EIF2AK4 variants, allowing for more accurate patient stratification .

• Study tissue-specific manifestations: Immunohistochemistry with EIF2AK4 antibodies is being used to examine expression patterns in pulmonary vascular tissues from PAH patients.

These applications highlight the evolving role of EIF2AK4 antibodies in translational research aimed at improving diagnosis and treatment of PAH-related conditions.

What emerging applications exist for EIF2AK4 antibodies in studying cellular stress responses?

Emerging applications for EIF2AK4 antibodies in studying cellular stress responses include:

• Single-cell analysis: EIF2AK4 antibodies are being adapted for use in single-cell proteomics and imaging to understand cell-to-cell variability in stress responses.

• Infection and immunity: Following discoveries about EIF2AK4's role in autophagy induction during bacterial infection, antibodies are being used to track activation patterns during various pathogenic challenges .

• Integrated multi-omics approaches: EIF2AK4 antibodies are combined with transcriptomics and metabolomics to create comprehensive models of cellular stress response networks.

• Tissue microenvironment studies: Spatial transcriptomics and proteomics approaches are incorporating EIF2AK4 antibodies to map stress responses within complex tissue architectures.

• Drug discovery platforms: High-content screening approaches utilizing EIF2AK4 antibodies are being developed to identify compounds that modulate integrated stress response pathways, with particular interest in the paradoxical activation of hypomorphic variants by certain inhibitors .

These emerging applications demonstrate the expanding utility of EIF2AK4 antibodies beyond traditional research methods, positioning them as valuable tools in addressing complex biological questions related to cellular stress responses.

How do results from antibody-based detection of EIF2AK4 compare with genetic and transcriptomic approaches?

When investigating EIF2AK4 in research settings, different methodological approaches provide complementary information:

What considerations should researchers make when choosing between different EIF2AK4 antibodies for specific research applications?

When selecting an EIF2AK4 antibody for research, consider these critical factors:

• Epitope location: Different antibodies recognize distinct epitopes of EIF2AK4. For variant studies, choose antibodies whose epitopes are not affected by the variants being studied. For phosphorylation studies, select phospho-specific antibodies targeting relevant sites.

• Application compatibility: Verify the antibody has been validated for your specific application (Western blot, IHC, IF, ELISA, ChIP). For example, the 11174-1-AP antibody has been validated for IHC and ELISA applications .

• Species reactivity: Confirm cross-reactivity with your model system. The 11174-1-AP antibody shows reactivity with human, mouse, and rat samples .

• Antibody type: Polyclonal antibodies (like 11174-1-AP) often provide higher sensitivity but may have batch-to-batch variation. Monoclonal antibodies offer greater specificity and consistency.

• Validation data: Review published literature and manufacturer data demonstrating specificity, particularly in knockout/knockdown systems.

• Functional validation: For studies of EIF2AK4 variants, consider whether antibodies can distinguish between functional and non-functional protein forms, which may require additional functional assays as demonstrated in recent research .