TRAPPC9 Antibody

What is TRAPPC9 and what are its key biological functions?

TRAPPC9 (Trafficking Protein Particle Complex 9), also known as NIBP (NIK and IKK-β-binding protein), is a subunit of the multiprotein TRAPP complex that functions as a guanine nucleotide exchange factor (GEF) for Rab proteins . This protein plays several critical roles:

• Vesicular trafficking: Functions in vesicular transport from endoplasmic reticulum to Golgi

• NF-κB signaling: Activates NF-κB through increased phosphorylation of the IKK complex

• Neuronal development: Essential for neurite elongation and branching processes

• Metabolic regulation: Involved in systemic glucose homeostasis and fat metabolism

TRAPPC9 is particularly abundant in postmitotic neurons of the cerebral cortex, hippocampus, and deep gray matter, with expression increasing during development .

What genetic disorders are associated with TRAPPC9 mutations?

Mutations in TRAPPC9 cause a rare genetic disorder now termed "NIBP syndrome" or "Intellectual developmental disorder, autosomal recessive 13" (OMIM #613192) . Clinical manifestations include:

Importantly, TRAPPC9 mutations are part of emerging disorders called "TRAPPopathies," characterized by overlapping neurodevelopmental phenotypes . TRAPPC9 variants have also been implicated in autism spectrum disorder, schizophrenia, and attention deficit/hyperactivity disorder .

What are the common applications for TRAPPC9 antibodies in neuroscience research?

TRAPPC9 antibodies serve multiple research purposes in neurodevelopmental studies:

These applications have been instrumental in elucidating TRAPPC9's role in neuronal development, intracellular trafficking, and neurodevelopmental disorders .

What considerations are important when designing Western blot experiments with TRAPPC9 antibodies?

Western blot analysis of TRAPPC9 requires special consideration due to its high molecular weight and post-translational modifications:

• Expected molecular weight discrepancy: Although the calculated molecular weight of TRAPPC9 is approximately 139 kDa, it typically runs at a significantly higher apparent molecular mass (128-140 kDa or even ~250 kDa) due to extensive post-translational modifications .

• Sample preparation protocol:

  • Use fresh tissue/cells whenever possible

  • Include protease inhibitors in lysis buffer

  • For brain tissue, use a protocol optimized for membrane-associated proteins

• Recommended positive controls: Mouse brain tissue , human brain tissue , and HEK-293 cells for in vitro studies .

• Gel concentration: Use low percentage (6-8%) gels to properly resolve this high molecular weight protein.

• Transfer conditions: Extend transfer time (overnight at low voltage) for complete transfer of high molecular weight proteins.

How should TRAPPC9 antibodies be optimized for immunohistochemistry of neural tissues?

Successful TRAPPC9 immunohistochemistry in neural tissues requires:

• Antigen retrieval method: Research indicates optimal results with TE buffer at pH 9.0; alternatively, citrate buffer at pH 6.0 can be used .

• Fixation considerations:

  • For perfusion-fixed tissues: 4% paraformaldehyde is recommended

  • For paraffin sections: Standard formalin fixation works well

• Blocking protocol:

  • Use 5-10% normal serum from the same species as the secondary antibody

  • Add 0.1-0.3% Triton X-100 for improved penetration in neural tissues

• Antibody incubation:

  • Primary antibody dilution: Start with 1:50-1:500 and optimize

  • Incubation time: Overnight at 4°C for best signal-to-noise ratio

• Signal detection systems:

  • For fluorescence: Secondary antibodies with minimal cross-reactivity

  • For chromogenic detection: DAB or similar systems with hematoxylin counterstain

How can TRAPPC9 antibodies help elucidate mechanisms of neurodevelopmental disorders?

TRAPPC9 antibodies have enabled several crucial discoveries regarding neurodevelopmental mechanisms:

• Cellular basis of microcephaly:

  • Immunostaining with anti-neurofilament heavy chain (NFH) and TRAPPC9 antibodies revealed decreased thickness of corpus callosum and cerebral cortex in Trappc9-deficient mice

  • Western blot analysis showed decreased NFH and MBP protein expression, suggesting reduced white matter as the primary cause of microcephaly

• Neurite development defects:

  • TRAPPC9 antibodies demonstrated that Trappc9 deficiency specifically impairs neurite elongation and branching, not neurite initiation or neural cell type specification

  • This finding established the specific developmental stage affected by TRAPPC9 mutations

• Distinguishing primary from secondary effects:

  • Ultrastructural and immunofluorescence studies using TRAPPC9 antibodies revealed that myelin structure remained normal in Trappc9-deficient mice, indicating reduced myelination is secondary to axonal defects

These findings provide a cellular and molecular framework for understanding intellectual disability in NIBP syndrome patients.

What insights have TRAPPC9 antibodies provided about dopaminergic signaling in intellectual disability?

TRAPPC9 antibodies have revealed an unexpected connection between TRAPPC9 deficiency and dopaminergic signaling:

• Altered dopamine receptor balance:

  • Western blot analyses using TRAPPC9 and dopamine receptor antibodies showed an imbalance between D1 and D2 receptor-containing neurons in Trappc9-deficient mice brains

  • This imbalance varied by brain region:

    • In striatum: Increased D1R:D2R ratio

    • In hippocampus: Decreased D1R:D2R ratio (0.232±0.0222 in wildtype vs. 0.122±0.0155 in knockouts)

• Pharmacological rescue of cognitive deficits:

  • Based on antibody-detected receptor imbalances, researchers tested D1 antagonist SCH23390 and D2 agonist quinpirole

  • Combined treatment restored exploratory activities of Trappc9-deficient mice to wild-type levels within 24 hours

  • This demonstrates a potential therapeutic approach for TRAPPC9-linked intellectual disability

• Dopamine secretion mechanisms:

  • Transcriptomic and proteomic analyses guided by TRAPPC9 antibody validation revealed impairments in dopamine secretion in Trappc9-deficient mice

  • Brain examinations showed normal dopamine synthesis but fewer release structures in dopamine-secreting neurons

This research establishes dopaminergic signaling as a key mechanism and potential therapeutic target in TRAPPC9-related intellectual disability.

How can TRAPPC9 antibodies illuminate the connections between intellectual disability and metabolic disorders?

TRAPPC9 antibodies have helped establish unexpected connections between intellectual disability and metabolic regulation:

• Obesity development mechanisms:

  • Antibody-based studies confirmed that Trappc9-deficient mice develop significant obesity shortly after weaning

  • Male Trappc9-deficient mice displayed infertility in addition to metabolic issues

• Non-alcoholic fatty liver disease (NAFLD):

  • TRAPPC9 antibodies helped identify that Trappc9 deficiency triggers both obesity and NAFLD

  • The same dopaminergic imbalance implicated in cognitive deficits also affects metabolic regulation

• Therapeutic targeting:

  • Based on antibody-identified pathways, chronic treatment combining D1R suppressors and D2R stimulators restored:

    • Systemic glucose homeostasis

    • Normal body weight regulation

    • Reduced lipid accumulation in adipose and liver tissues

This research suggests TRAPPC9's dual role in neurodevelopmental and metabolic regulation, potentially through shared dopaminergic mechanisms.

Why might Western blot results show variable molecular weights for TRAPPC9?

TRAPPC9 antibodies frequently detect bands at molecular weights different from the calculated 139 kDa:

• Post-translational modifications:

  • Patient-derived cell lines analyzed with TRAPPC9 antibodies revealed bands at ~250 kDa rather than the predicted ~140 kDa

  • This suggests extensive post-translational modification of TRAPPC9 protein

• Splice variants:

  • According to RefSeq, there are two transcript variants of TRAPPC9 in humans:

    • Variant 1 (longer): 1246 amino acids

    • Variant 2 (shorter): 1148 amino acids

  • Antibodies may detect one or both variants depending on the epitope

• Recommended troubleshooting approach:

  • Run positive control samples (brain tissue) alongside experimental samples

  • Consider using antibodies targeting different epitopes to confirm findings

  • When possible, include knockout/knockdown controls to verify specificity

How can researchers resolve inconsistent subcellular localization patterns in TRAPPC9 immunostaining?

Inconsistent subcellular localization in TRAPPC9 immunostaining may reflect biological complexity rather than technical issues:

• Expected localization patterns:

  • Primary localization: Cytoplasm

  • Secondary localizations: Endoplasmic reticulum, Golgi apparatus, cis-Golgi network

  • No evidence of nuclear localization

• Confocal microscopy findings:

  • High-magnification and confocal imaging shows TRAPPC9 immunoreactivity is widespread in the cytoplasm

  • No specific co-localization with Golgi apparatus, late endosomes, or early endosomes markers

• Technical recommendations:

  • Use multiple antibodies targeting different epitopes

  • Employ co-staining with organelle markers (GM130 for Golgi, calnexin for ER)

  • Consider super-resolution microscopy for more detailed localization

  • Include appropriate controls (knockout tissues or siRNA-treated cells)

• Functional context:

  • Consider that TRAPPC9's role in vesicular trafficking may result in dynamic localization patterns that vary with cellular state

What approaches can resolve contradictory findings between mouse and human TRAPPC9 studies?

Research has revealed both similarities and differences between mouse and human TRAPPC9:

How might TRAPPC9 antibodies facilitate development of therapeutic approaches for TRAPPopathies?

Emerging research suggests several promising therapeutic strategies that will require TRAPPC9 antibodies for development and validation:

• Dopaminergic modulation therapy:

  • Antibody-based studies identified dopamine receptor imbalance as a key mechanism

  • Pharmacological combination of D1R antagonists and D2R agonists shows promise for:

    • Improving cognitive performance

    • Restoring metabolic homeostasis

  • Future directions: Development of targeted therapeutics with fewer side effects

• TRAPPII complex targeting:

  • Research highlights "the importance of developing therapeutic approaches targeting the TRAPPII complex to cure TRAPPopathies"

  • TRAPPC9 antibodies will be essential for:

    • Screening potential compounds

    • Validating target engagement

    • Monitoring restoration of normal trafficking

• Neurite development enhancement:

  • Since antibody studies showed that Trappc9 deficiency specifically impairs neurite elongation and branching

  • Therapeutic strategies promoting these processes could mitigate developmental deficits

  • TRAPPC9 antibodies would be crucial for measuring treatment effectiveness

What novel techniques can enhance TRAPPC9 antibody applications in neurodevelopmental research?

Advanced technologies can expand the utility of TRAPPC9 antibodies:

• Proximity labeling approaches:

  • BioID or APEX2 fusions with TRAPPC9 coupled with antibody detection

  • Would reveal transient interacting partners in different neural cell types

  • Could identify cell-type specific functions of TRAPPC9

• Super-resolution microscopy:

  • Combining TRAPPC9 antibodies with techniques like STORM or STED

  • Would provide nanometer-scale resolution of TRAPPC9 localization

  • Could reveal previously undetected subcellular organization

• Live-cell imaging technologies:

  • Developing nanobodies based on existing TRAPPC9 antibodies

  • Would enable real-time visualization of TRAPPC9 dynamics

  • Could reveal trafficking mechanisms in living neurons

• Spatial transcriptomics integration:

  • Combining TRAPPC9 antibody staining with spatial transcriptomics

  • Would correlate protein localization with gene expression patterns

  • Could identify regional vulnerabilities in development

These advanced applications will require careful validation but offer tremendous potential for understanding TRAPPC9's complex roles in neurodevelopment.