HSPA9 Antibody

What is HSPA9 and what cellular compartments does it localize to?

HSPA9 is a 75 kDa member of the heat shock protein 70 family. Although primarily localized to the mitochondrial matrix, HSPA9 is also found in the endoplasmic reticulum, plasma membrane, and cytoplasmic vesicles . It functions as a heat-shock cognate protein involved in multiple cellular processes including protein folding, translocation of nuclear and mitochondrial polypeptides, stress response, and maintenance of mitochondrial function .

Unlike other HSP70 family members (HSP70, HSC70), GRP75 (HSPA9) and GRP78 are unresponsive to heat stress but are induced by glucose deprivation . This distinguishing characteristic makes HSPA9 an interesting target for metabolic and stress-related studies.

Which applications are HSPA9 antibodies validated for?

HSPA9 antibodies have been validated for multiple experimental applications with specific dilution recommendations:

Researchers should always validate the antibody in their specific experimental system as optimal dilutions can be sample-dependent .

How does HSPA9 function relate to mitochondrial dynamics and what methods best reveal this?

HSPA9 plays a crucial role in mitochondrial protein import and quality control. Research has shown that HSPA9 deficiency disrupts mitochondrial dynamics, specifically increasing mitochondrial fission and reactive oxygen species (ROS) production .

Methodological approaches:

• ROS measurement: Use mitochondria-targeted redox-sensitive fluorescent sensors like HyPer-PTS1 to detect hydrogen peroxide specifically in peroxisomes following HSPA9 manipulation

• Mitochondrial morphology assessment: Fluorescence microscopy with mitochondrial markers (e.g., TOMM20) to evaluate fission/fusion dynamics after HSPA9 knockdown

• Protein interaction studies: Co-IP followed by mass spectrometry to identify HSPA9 interaction partners within the mitochondrial import machinery

Research has demonstrated that HSPA9 interacts with critical components of the MINOS/MitOS complex, including IMMT and CHCHD3, which associates with outer membrane proteins like SAMM50, MTX1, and MTX2 .

What is the relationship between HSPA9 and disease pathogenesis?

HSPA9 has been implicated in multiple disease processes through various mechanisms:

• Inflammatory Bowel Disease: HSPA9 downregulation exacerbates colitis symptoms through increased immune cell infiltration, elevated proinflammatory cytokines, decreased tight junctions, and altered macrophage polarization

• Neurodegenerative Diseases: HSPA9 mutants found in Parkinson's disease fail to rescue peroxisome loss, suggesting a potential pathogenic mechanism involving impaired protein quality control

• Cancer: HSPA9 functions in the control of cell proliferation and has been identified as part of the MEK1/2 complex, potentially regulating Raf/MEK/ERK tumor-suppressive signaling

• Glioblastoma: Research has identified an OMA1/HSPA9/cGAS/PD-L1 axis where OMA1 competitively binds to HSPA9 to induce mitophagy and promote immune evasion

• Hematological Disorders: HSPA9 is associated with autosomal dominant sideroblastic anemia and has been recognized in schizophrenia-associated genetic loci

What considerations are important for optimizing Western blot detection of HSPA9?

For optimal Western blot results with HSPA9 antibodies:

• Expected band size: ~75 kDa (73,680 Da precise molecular weight)

• Positive controls: HeLa cell lysate (heat shocked), HepG2, A549, Jurkat, and NIH/3T3 cell lysates have all shown consistent HSPA9 detection

• Buffer conditions: Some antibodies work optimally with specific buffer systems - for example, certain HSPA9 antibodies perform best with Immunoblot Buffer Group 2

• Detection sensitivity: Some HSPA9 antibodies show exceptionally high sensitivity, allowing dilutions up to 1:50000, enabling economical use in large-scale experiments

• Secondary antibody selection: For polyclonal antibodies raised in rabbit, anti-rabbit HRP or fluorescently labeled secondaries work well; for monoclonal antibodies (e.g., clone 30A5 or 10D7), species-appropriate secondaries must be selected

How can I address inconsistent HSPA9 antibody results across different experimental platforms?

When encountering inconsistent results with HSPA9 antibodies:

• Epitope accessibility: HSPA9 functions in multiple cellular compartments, and epitope accessibility may vary by fixation/permeabilization method. For mitochondrial detection, ensure appropriate permeabilization

• Antibody specificity validation: Use HSPA9-depleted cells as negative controls or blocking peptides to confirm specificity. Some antibodies have been specifically validated with antigen peptide competition tests

• Buffer compatibility: HSPA9 antibody performance may vary with buffer conditions - test multiple buffer systems particularly when switching between applications

• Post-translational modifications: HSPA9 undergoes various modifications that might affect antibody recognition. Use antibodies targeting unmodified regions when possible

• Cross-reactivity: Verify species cross-reactivity experimentally. While some HSPA9 antibodies work across human, mouse, and rat samples, others have limited species reactivity

How does HSPA9 contribute to peroxisomal homeostasis and what methods reveal this function?

Research has identified HSPA9 as a novel pexophagy regulator:

• Peroxisome visualization: Studies have used peroxisome-targeted fluorescent proteins (e.g., turquoise2-PTS1) in HeLa cells to screen for peroxisomal regulators, identifying HSPA9 as a potent regulator of pexophagy

• Mechanism: HSPA9 depletion increases peroxisomal reactive oxygen species (ROS), which can be measured using peroxisome-targeted HyPer, a genetically modified redox-sensitive fluorescent sensor protein

• ROS scavenging experiments: The connection between HSPA9, ROS, and peroxisomal degradation has been demonstrated using N-acetylcysteine (NAC) treatment, which blocks pexophagy in HSPA9-depleted cells

• Genetic validation: HSPA9 function in peroxisome maintenance has been confirmed across species, as knockdown of Hsc70-5 (HSPA9 ortholog) in Drosophila decreases peroxisomes, while reconstitution with wild-type HSPA9 rescues this phenotype

How does HSPA9 relate to reproductive biology and what methodologies reveal this function?

HSPA9 has been implicated in sperm quality and male fertility:

• Differential expression analysis: Proteomics comparison between high and low-motility spermatozoa from fertile men revealed HSPA9 is significantly decreased in low-motility sperm

• Subcellular localization: Immunofluorescence studies show HSPA9 is mainly expressed on the acrosome and sperm tail, suggesting potential roles in sperm function

• Functional validation: Antibody blocking experiments with HSPA9 antibodies showed no significant effect on sperm motility (unlike HSPA1L), providing important negative data about its direct role in motility

• Developmental context: Comparison of young adult and elderly testes showed decreased HSPA9 expression in elderly testes characterized by poor spermatogenesis, suggesting HSPA9 may be involved in maintaining spermatogenesis quality

How is HSPA9 involved in immune regulation and cancer immunotherapy?

Recent research has uncovered HSPA9's role in immune checkpoint regulation:

• OMA1/HSPA9/cGAS/PD-L1 axis: In glioblastoma, OMA1 competitively binds to HSPA9 to induce mitophagy and promote immune evasion

• Mechanism: This interaction promotes PD-L1 levels in GBM cells through a pathway involving:

  • OMA1 binding to HSPA9

  • Induction of mitophagy

  • Increased mitochondrial DNA release

  • Activation of cGAS-STING pathway

  • Enhanced PD-L1 transcription

  • Immune evasion through PD-1 binding and T cell cytotoxicity inhibition

• Therapeutic implications: This newly identified axis represents a potential therapeutic target for immunotherapy in glioblastoma, particularly for improving response to checkpoint inhibitors

What experimental considerations are important when studying HSPA9's role in neurodegenerative diseases?

When investigating HSPA9 in neurodegenerative contexts:

• Disease-specific mutations: HSPA9 mutations found in Parkinson's disease failed to rescue peroxisome loss in both cellular and Drosophila models, suggesting a mechanistic connection

• Mitochondrial dynamics: Since HSPA9 regulates mitochondrial function, assess mitochondrial morphology, function, and ROS production using appropriate markers and fluorescent probes in neuronal models

• Peroxisomal degradation: Monitor peroxisome numbers in neurodegenerative disease models with HSPA9 manipulation using peroxisomal markers like ABCD3 and CAT (catalase)

• Rescue experiments: Test if wild-type HSPA9 can rescue neurodegeneration phenotypes that disease-associated mutants cannot, providing evidence for pathological mechanisms

• Stress responses: As HSPA9 is involved in stress response, examine how oxidative or proteotoxic stress affects HSPA9 function in neuronal contexts through various cellular stress assays