TMEM184B Antibody

What is TMEM184B and why is it significant in neurological research?

TMEM184B is an evolutionarily conserved seven-pass transmembrane protein highly expressed in the nervous system. It has emerged as a critical regulator of endolysosomal acidification in neurons, making it significant for understanding both neurodevelopmental and neurodegenerative disorders. Recent research has identified TMEM184B as a novel regulator that interacts with vacuolar ATPase (V-ATPase) components to promote assembly of V0 and V1 subdomains, which facilitates proper lumenal acidification .

Sequence variations in TMEM184B have been linked to neurodevelopmental disorders, suggesting its importance in proper nervous system development and function. Additionally, TMEM184B localizes to early and late endosomes, further supporting its role in the endosomal system and neuronal protein homeostasis .

How do I select the appropriate TMEM184B antibody for my specific research application?

Selection should be guided by your experimental requirements:

• Application compatibility: Different antibodies are validated for specific applications. For instance, antibody ABIN2782777 is validated for Western Blotting , while the 28031-1-AP antibody is validated for IHC and ELISA applications .

• Species reactivity: Consider the origin of your experimental tissue. Antibody ABIN2782777 demonstrates broad cross-reactivity across multiple species including human, mouse, rat, cow, guinea pig, horse, rabbit, dog, and zebrafish , while some antibodies like 28031-1-AP may have more limited reactivity (e.g., human samples) .

• Epitope recognition: Targeting specific regions of TMEM184B may be important. For example, ABIN2782777 targets the middle region with the sequence "STVILQAFGK YRDGDFDVTS GYLYVTIIYN ISVSLALYAL FLFYFATREL" .

• Conjugation requirements: Determine whether unconjugated or conjugated (FITC, biotin, etc.) antibodies are needed based on your detection system .

What are the recommended protocols for antigen retrieval when performing IHC with TMEM184B antibodies?

Antigen retrieval is critical for successful immunohistochemistry with TMEM184B antibodies. The following protocols have been validated:

• Primary recommendation: Tris-EDTA (TE) buffer at pH 9.0 . This appears to provide optimal epitope exposure for TMEM184B detection in fixed tissues.

• Alternative approach: Citrate buffer at pH 6.0 . This may be used if TE buffer yields suboptimal results.

For neuronal tissues specifically, which express high levels of TMEM184B, optimal dilution ranges between 1:250-1:1000 for IHC applications . When working with mouse brain or cerebellum tissue samples, careful optimization of these protocols is particularly important due to the complex tissue architecture and varying expression levels of TMEM184B throughout different brain regions.

How should I design RNA interference experiments to effectively knockdown TMEM184B expression?

Effective TMEM184B knockdown requires careful consideration of the following elements:

Vector selection and design:

• Lentiviral vector systems like GV115 have been successfully used to deliver shRNAs targeting TMEM184B .

• Include appropriate negative controls using scrambled sequences (e.g., TTCTCCGAACGTGTCACGT) to control for non-specific effects .

Transfection and validation protocol:

• Co-transfect your constructed vectors into HEK-293T cells with lentiviral packaging vectors using Lipofectamine 2000 or similar reagents.

• Collect viruses 48 hours post-transfection.

• Infect target cells (e.g., FaDu cells for HPSCC studies).

• Rigorously validate knockdown efficiency using both RT-qPCR and Western blotting to confirm both mRNA and protein reduction .

Experimental timeline:

• Perform downstream assays approximately 48 hours after infection to achieve optimal knockdown while minimizing compensatory cellular responses .

• For longer studies, stable cell lines expressing the shRNA constructs should be established and periodically validated for maintained knockdown.

What methodological approaches should be used to accurately assess endolysosomal acidification changes in TMEM184B-deficient neurons?

Assessing endolysosomal acidification in TMEM184B-deficient neurons requires specialized techniques:

Preparation of neuronal cultures:

• Use primary mouse cortical neurons, as they have demonstrated reliable responses in TMEM184B studies .

• Ensure experimental design includes appropriate wild-type controls cultured under identical conditions.

pH measurement techniques:

• pH-sensitive fluorescent probes that accumulate in acidic compartments

• Ratiometric imaging to quantify pH differences between control and TMEM184B-deficient neurons

• Live-cell imaging to track dynamic changes in endolysosomal pH

V-ATPase assembly assessment:

• Biochemical fractionation followed by co-immunoprecipitation to evaluate V0 and V1 subdomain assembly states

• Western blotting to quantify the relative proportions of assembled vs. disassembled V-ATPase complexes

These approaches have revealed that loss of TMEM184B results in significant reductions in endolysosomal acidification, associated with impaired assembly of the V-ATPase V0 and V1 subcomplexes .

What controls are essential when investigating TMEM184B expression in tumor versus normal tissues?

When comparing TMEM184B expression between tumor and normal tissues, implement these critical controls:

Tissue sampling controls:

• Use paired tumor and adjacent normal tissues from the same patients whenever possible

• Ensure tissue sections are matched for cellular composition and processing

Immunohistochemistry validation:

• Include both positive and negative tissue controls in each staining batch

• Verify antibody specificity using TMEM184B-knockdown tissues or cells as negative controls

• Use standardized scoring systems (e.g., intensity scoring on a 0-3 scale) for quantitative comparisons

Bioinformatics verification:

• Correlate IHC findings with mRNA expression data from public databases like TCGA and GEO

• For HPSCC specifically, datasets like GSE58911 can provide additional validation

This multi-platform approach has demonstrated significantly higher TMEM184B expression in hypopharyngeal squamous cell carcinoma tissues compared to adjacent normal mucosa, which has been further validated through bioinformatic analysis of public datasets .

How can I investigate the precise mechanism by which TMEM184B regulates V-ATPase assembly in neurons?

Investigating this mechanism requires sophisticated approaches:

Protein interaction mapping:

• Perform proteomic analysis of TMEM184B-interacting proteins in neuronal cells

• Use proximity labeling methods (BioID or APEX) to identify transient interactions

• Confirm direct interactions with co-immunoprecipitation followed by Western blotting

Structure-function analysis:

• Create domain-specific mutants of TMEM184B to identify regions critical for V-ATPase interaction

• Assess the impact of these mutations on V-ATPase assembly and endolysosomal acidification

Subcellular localization studies:

• Use super-resolution microscopy to precisely map TMEM184B location within the endolysosomal system

• Perform time-lapse imaging to track dynamic relationships between TMEM184B and V-ATPase components

This approach has revealed that TMEM184B shows enriched interaction with components involved in endosomal trafficking and function, particularly the V-ATPase complex. Loss of TMEM184B impairs assembly of V-ATPase V0 and V1 subcomplexes, suggesting a mechanistic basis for disrupted neuronal function in TMEM184B-associated disorders .

What are the best approaches to reconcile contradictory findings regarding TMEM184B function between cancer and neurological research?

Research reveals TMEM184B has context-dependent functions that require integrated analysis:

Comparative analysis strategies:

• Perform parallel experiments in neuronal and cancer cell models using identical methods

• Compare protein interaction networks between cell types to identify shared versus unique binding partners

• Investigate whether cell-type specific post-translational modifications alter TMEM184B function

Potential explanations for divergent findings:

• In cancer contexts (e.g., HPSCC), TMEM184B promotes cell proliferation and inhibits apoptosis

• In neuronal contexts, TMEM184B regulates endolysosomal acidification and synaptic function

• These differences may reflect tissue-specific regulatory networks or alternative splicing

Resolution approaches:

• Examine whether endolysosomal acidification is also altered in TMEM184B-overexpressing cancer cells

• Investigate if neuronal phenotypes include proliferation changes similar to cancer models

• Consider evolutionary analysis of TMEM184B function across species to identify core versus specialized functions

What troubleshooting approaches should be employed when TMEM184B antibodies show inconsistent staining patterns?

When encountering inconsistent TMEM184B antibody staining, systematically address these factors:

Antibody validation:

• Confirm antibody specificity using TMEM184B-knockout or knockdown samples

• Test multiple antibodies targeting different epitopes of TMEM184B

• Validate results with orthogonal methods (e.g., in situ hybridization for mRNA)

Sample preparation optimization:

• Fixation: Test multiple fixation methods and durations

• Antigen retrieval: Compare TE buffer (pH 9.0) versus citrate buffer (pH 6.0)

• Blocking: Optimize blocking solutions to reduce background

Application-specific adjustments:

• For IHC: Titrate antibody concentrations (1:250-1:1000 range recommended)

• For Western blotting: Adjust protein loading, transfer conditions, and detection methods

• For immunofluorescence: Consider signal amplification methods

Experimental controls table:

How can TMEM184B research inform therapeutic approaches for neurodevelopmental disorders?

TMEM184B's role in neuronal function suggests several therapeutic avenues:

Potential intervention strategies:

• Target V-ATPase assembly to rescue endolysosomal acidification defects in TMEM184B-deficient neurons

• Develop small molecules that mimic TMEM184B's interaction with V-ATPase components

• Explore gene therapy approaches to restore functional TMEM184B in affected neurons

Biomarker development:

• Investigate whether CSF or plasma markers reflect TMEM184B dysfunction in neurodevelopmental disorders

• Develop imaging probes to monitor endolysosomal acidification in vivo

Disease modeling:

• Generate patient-derived iPSCs carrying TMEM184B variants to create relevant neuronal models

• Use CRISPR-engineered animal models to understand systemic effects of TMEM184B dysfunction

The identification of TMEM184B as a regulator of endolysosomal acidification provides mechanistic insight into its link to neurological disorders and suggests that enhancing this pathway could improve outcomes in diseases involving lysosomal dysfunction .

What are the most promising techniques for studying TMEM184B involvement in cancer progression and metastasis?

Several sophisticated approaches can advance understanding of TMEM184B in cancer:

Advanced in vitro models:

• 3D organoid cultures to better recapitulate tumor microenvironment

• Co-culture systems with stromal components to assess tumor-stroma interactions

• Microfluidic devices to study invasion and migration in controlled gradients

In vivo metastasis models:

• Orthotopic xenograft models with TMEM184B-modified cancer cells

• Intravital imaging to track cancer cell behavior in live animals

• Circulating tumor cell isolation and characterization

Multi-omics integration:

• Correlate TMEM184B expression with cancer genomic, transcriptomic, and proteomic profiles

• Network analysis to identify TMEM184B-associated pathways in different cancer types

TMEM184B has demonstrated oncogenic functions in hypopharyngeal squamous cell carcinoma, promoting cell growth, invasion, and migration while inhibiting apoptosis . These findings suggest TMEM184B could serve as both a diagnostic biomarker and therapeutic target for HPSCC.

How might the dual roles of TMEM184B in endolysosomal function and tumor biology be mechanistically connected?

The dual functionality of TMEM184B suggests intriguing mechanistic connections:

Hypothesized mechanistic links:

• Altered endolysosomal pH may affect receptor recycling and signaling pathway activation in cancer cells

• Impaired degradation of growth factor receptors due to endolysosomal dysfunction could promote sustained proliferative signaling

• Changes in autophagy efficiency resulting from endolysosomal acidification defects might impact cancer cell survival under stress

Experimental approaches to test these hypotheses:

• Compare endolysosomal pH and V-ATPase assembly in normal versus TMEM184B-overexpressing cancer cells

• Track trafficking and degradation of growth factor receptors in cells with modified TMEM184B levels

• Assess autophagy flux and stress responses in these cellular models

Potential unifying model: TMEM184B may function as a molecular switch that coordinates membrane trafficking, signaling receptor degradation, and cellular metabolism through its effects on the endolysosomal system. In neurons, this primarily affects proteostasis and synaptic function, while in cancer cells, these same mechanisms might be co-opted to promote survival and proliferation.