TMEM237 Antibody

What is TMEM237 and why is it important in scientific research?

TMEM237 (Transmembrane Protein 237, also known as ALS2CR4 or JBTS14) is a tetraspanin protein that localizes to the ciliary transition zone (TZ) and plays a crucial role in ciliogenesis. Its significance in research stems from its involvement in multiple biological processes and disease states:

• Ciliopathy research: Mutations in TMEM237 cause Joubert syndrome-14, a ciliopathy characterized by brain malformations and various developmental abnormalities .

• Signaling pathway studies: TMEM237 is involved in Wnt signaling regulation .

• Cancer research: TMEM237 is overexpressed in hepatocellular carcinoma (HCC) and promotes tumor progression through interaction with the Pyk2/ERK pathway .

• Developmental biology: TMEM237 is important for proper ciliogenesis in mammalian cells, zebrafish, and C. elegans .

Understanding TMEM237 function provides insights into fundamental cellular processes and potential therapeutic targets for associated diseases.

What are the key considerations when selecting a TMEM237 antibody for research?

When selecting a TMEM237 antibody, researchers should consider:

Epitope recognition: Different antibodies recognize distinct regions of TMEM237. For example:

• Antibodies targeting amino acids 181-230

• Antibodies targeting amino acids 59-78

• Antibodies targeting amino acids 251-300

• Antibodies targeting V165-R215

• Antibodies targeting the sequence ELQYANELGVEDEDIITDEQTTVEQQSVFTAPTGISQPVGKVFVEKSRRFQAADRSELIKTTENIDVSMDVKP

Validated applications: Ensure the antibody is validated for your specific application:

• Western blotting (typical working dilutions: 0.04-0.4 μg/mL)

• Immunohistochemistry (typical working dilutions: 1:500-1:1000)

• ELISA

• Immunocytochemistry/Immunofluorescence

Species reactivity: Confirm reactivity with your experimental model:

• Human-reactive antibodies

• Mouse-reactive antibodies

• Some antibodies also react with cow, dog, horse, pig, rabbit, and rat

Validation status: Look for antibodies with enhanced validation using orthogonal methods or independent validation .

What is the optimal protocol for detecting TMEM237 localization in ciliated cells?

To effectively detect TMEM237 localization in ciliated cells:

Sample preparation:

• Culture ciliated cells on coverslips until they reach appropriate confluence

• Induce ciliogenesis by serum starvation (24-48 hours)

• Fix cells with 4% paraformaldehyde for 10 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 for 5 minutes

Immunostaining protocol:

• Block with 5% BSA for 1 hour at room temperature

• Incubate with primary anti-TMEM237 antibody (1:500-1:1000 dilution) overnight at 4°C

• Wash 3 times with PBS

• Incubate with appropriate secondary antibody (typically Alexa-Fluor 488 or 568-conjugated goat anti-rabbit IgG) for 1 hour at room temperature

• Counterstain cilia with anti-polyglutamylated tubulin (GT-335) to mark the axoneme

• Counterstain nuclei with DAPI

• Mount slides with appropriate mounting medium

Imaging considerations:

• Use confocal microscopy for optimal visualization of the ciliary transition zone

• TMEM237 should co-localize with other transition zone markers

• Compare localization with known transition zone proteins like RPGRIP1L/MKS5

This protocol can be adapted for specific cell types including renal epithelial cells, fibroblasts, and neuronal cells that express primary cilia.

How can I optimize Western blotting conditions for TMEM237 detection?

For optimal Western blot detection of TMEM237:

Sample preparation:

• Prepare cell/tissue lysates in RIPA buffer supplemented with protease inhibitor cocktail

• Ensure complete lysis by sonication (3 × 10s pulses)

• Centrifuge at 14,000 × g for 15 minutes at 4°C to remove debris

• Quantify protein using Bradford or BCA assay

SDS-PAGE and transfer parameters:

• Load 20-50 μg of protein per lane

• Use 10-12% polyacrylamide gels (TMEM237 is approximately 45 kDa)

• Transfer to PVDF membrane at 100V for 90 minutes in cold transfer buffer with 20% methanol

Immunodetection optimization:

• Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Incubate with anti-TMEM237 antibody at 0.04-0.4 μg/mL dilution in 5% BSA/TBST overnight at 4°C

• Wash 3 × 10 minutes with TBST

• Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

• Wash 3 × 10 minutes with TBST

• Develop using ECL substrate and image

Validation controls:

• Use lysates from TMEM237 knockout or knockdown cells as negative controls

• Use TMEM237-overexpressing cells as positive controls

• Include β-actin as loading control

• For additional validation, pre-incubate antibody with immunizing peptide to confirm specificity

Note that TMEM237 may appear at different molecular weights depending on post-translational modifications and splice variants.

How should I design experiments to study TMEM237 interactions with transition zone proteins?

When designing experiments to study TMEM237 interactions with transition zone proteins:

Co-immunoprecipitation approach:

• Prepare cell lysates in mild lysis buffer (1% NP-40 or similar) to preserve protein-protein interactions

• Pre-clear lysates with protein A/G beads

• Immunoprecipitate with anti-TMEM237 antibody or anti-tag antibody for overexpressed constructs

• Analyze precipitates by Western blotting for potential interacting partners (NPHP1, NPHP4, MKS2, MKSR1/B9D1, MKSR2/B9D2, MKS5/RPGRIP1L)

• Perform reciprocal IPs to confirm interactions

• Include appropriate controls (IgG control, input samples)

Proximity ligation assay (PLA):

• Fix cells and perform standard immunofluorescence protocol with antibodies against TMEM237 and potential interacting partners

• Use species-specific PLA probes and detection reagents

• Analyze interaction by fluorescence microscopy, quantifying PLA signals per cell

Mass spectrometry identification of novel interactors:

• Perform immunoprecipitation with anti-TMEM237 antibody

• Run samples on SDS-PAGE and analyze by LC-MS/MS

• Identify potential interacting proteins through database searching

• Validate novel interactions by co-IP and functional assays

Functional validation of interactions:

• Design genetic interaction studies in model organisms (C. elegans, zebrafish)

• Create single and double mutants/knockdowns

• Analyze phenotypes for evidence of genetic interaction (synergistic effects)

• Perform rescue experiments with wild-type and mutant constructs

This multi-method approach ensures robust identification and characterization of genuine protein-protein interactions.

What are the key experimental controls needed when studying TMEM237 in ciliopathy models?

When studying TMEM237 in ciliopathy models, include these essential controls:

Genetic model validation controls:

• Confirm knockout/knockdown efficiency by qRT-PCR and Western blot

• Sequence verification of mutations in patient-derived cells or engineered models

• Include wild-type controls from matching genetic background

• For transgenic rescue, validate expression levels to ensure physiological relevance

Ciliogenesis phenotype controls:

• Quantify percentage of ciliated cells using axonemal markers (acetylated tubulin or polyglutamylated tubulin)

• Measure ciliary length in addition to presence/absence

• Compare to established ciliopathy gene mutants (e.g., other transition zone components)

• Include positive controls known to affect ciliogenesis (e.g., IFT88 depletion)

Signaling pathway analysis controls:

• Include positive and negative controls for Wnt pathway activation

• Test multiple readouts of pathway activity (e.g., TOPFlash reporter, β-catenin localization, target gene expression)

• Use pathway agonists and antagonists as reference points

Tissue-specific controls:

• For brain development studies: include markers for specific neuronal populations affected in Joubert syndrome

• For renal studies: include markers of renal epithelial cell polarity and tubule formation

Rescue experiment controls:

• Include both wild-type TMEM237 and disease-associated mutant forms

• Titrate expression levels to avoid overexpression artifacts

• Include domain deletion constructs to identify functional regions

These controls ensure experimental rigor and facilitate interpretation of results in the context of ciliopathy pathogenesis.

How can TMEM237 antibodies be utilized to investigate the role of TMEM237 in cancer progression?

TMEM237 antibodies can be strategically employed to investigate its role in cancer progression through multiple approaches:

Expression profiling in cancer tissues:

• Perform immunohistochemistry on tissue microarrays containing tumor and matched normal tissues

• Score TMEM237 expression levels (0-3 scale for intensity, 0-4 scale for percentage of positive cells)

• Calculate IHC scores by multiplying intensity and percentage scores

• Correlate expression with clinical parameters, tumor stage, and patient survival

Mechanistic studies in cancer cell lines:

• Establish stable TMEM237 overexpression or knockdown cancer cell lines

• Analyze proliferation using CCK-8, colony formation, and EdU assays

• Assess migration and invasion using Transwell assays

• Examine EMT markers (E-cadherin, N-cadherin, vimentin) by immunoblotting with TMEM237 modulation

• Use immunofluorescence to study subcellular localization in cancer cells

Signaling pathway analysis:

• Perform co-immunoprecipitation to identify cancer-relevant interacting partners

• Analyze specific pathway activation (e.g., Pyk2/ERK1/2) using phospho-specific antibodies

• Conduct RNA-seq following TMEM237 modulation to identify regulatory networks

• Validate key targets using ChIP assays if transcriptional mechanisms are implicated

In vivo tumor models:

• Establish xenograft models using TMEM237-modified cancer cells

• Monitor tumor growth, metastasis, and EMT marker expression in vivo

• Perform IHC on xenograft sections for Ki67, E-cadherin, N-cadherin, and vimentin

• Use TMEM237 antibodies for ex vivo analysis of tumor tissues

This comprehensive approach can reveal how TMEM237 contributes to cancer hallmarks including proliferation, invasion, and metastasis.

What methodological approaches can be used to study the functional relationship between TMEM237 and its interaction partner NPHP1?

To investigate the functional relationship between TMEM237 and NPHP1:

Biochemical characterization of the interaction:

• Map binding domains through deletion constructs and co-IP experiments

• Determine binding affinities using purified proteins and surface plasmon resonance

• Investigate whether the interaction is direct or requires additional cofactors

• Assess whether disease-associated mutations affect the interaction

Subcellular localization studies:

• Perform immunofluorescence to determine if TMEM237 and NPHP1 co-localize at the transition zone

• Use super-resolution microscopy (STORM, PALM) for precise localization

• Test whether depletion of one protein affects localization of the other

• Determine the order of recruitment to the transition zone during ciliogenesis

Functional interdependence:

• Generate single and double knockdowns/knockouts of TMEM237 and NPHP1

• Compare phenotypes of single vs. double depletions to assess genetic interaction

• Perform rescue experiments with wild-type and mutant constructs

• Analyze downstream signaling effects (e.g., Pyk2/ERK phosphorylation)

Pathway analysis:

• Investigate whether TMEM237 affects NPHP1 interaction with Pyk2

• Analyze formation of multiprotein complexes through sequential IP experiments

• Determine the effects of TMEM237-NPHP1 interaction on Wnt signaling

• Identify common downstream targets through transcriptomics/proteomics approaches

Model organism studies:

• Create genetic models in zebrafish or C. elegans

• Analyze genetic interactions and cilia formation in vivo

• Test tissue-specific phenotypes relevant to ciliopathies

• Perform rescue experiments with human proteins to confirm conserved functions

These approaches provide a comprehensive understanding of how TMEM237 and NPHP1 functionally interact to regulate cellular processes.

What are common challenges in detecting TMEM237 by immunofluorescence and how can they be overcome?

Common challenges and solutions for TMEM237 immunofluorescence detection:

Low signal intensity:

• Challenge: TMEM237 is present at low levels or concentrated in the small transition zone region

• Solutions:

  • Optimize antibody concentration (try 1:100-1:1000 dilutions)

  • Extend primary antibody incubation to overnight at 4°C

  • Use signal amplification methods (e.g., tyramide signal amplification)

  • Try different fixation methods (PFA vs. methanol)

  • Use antigen retrieval techniques if necessary

High background:

• Challenge: Non-specific binding of antibodies

• Solutions:

  • Increase blocking time and concentration (5-10% BSA or normal serum)

  • Ensure thorough washing steps (3-5 washes, 5-10 minutes each)

  • Pre-absorb antibodies with cell lysates from TMEM237 knockout cells

  • Use highly cross-adsorbed secondary antibodies

  • Include 0.1-0.3% Triton X-100 in antibody dilution buffer

Co-localization difficulties:

• Challenge: Precisely localizing TMEM237 to the transition zone

• Solutions:

  • Use established transition zone markers (RPGRIP1L/MKS5) as co-staining references

  • Include ciliary axoneme markers (acetylated tubulin) for orientation

  • Use super-resolution microscopy techniques

  • Employ structured illumination microscopy (SIM) for improved resolution

Variability between samples:

• Challenge: Inconsistent ciliation or TMEM237 expression

• Solutions:

  • Standardize cell culture conditions and serum starvation protocols

  • Analyze sufficient numbers of cells (>100 per condition)

  • Use quantitative image analysis software for objective assessment

  • Include positive controls (cells known to express TMEM237)

Specificity concerns:

• Challenge: Confirming antibody specificity

• Solutions:

  • Include TMEM237 knockdown/knockout controls

  • Validate with multiple antibodies against different epitopes

  • Perform peptide competition assays

  • Use tagged TMEM237 constructs for validation

These approaches help ensure reliable and interpretable TMEM237 immunofluorescence data.

How can researchers accurately interpret contradictory data regarding TMEM237 function in different experimental systems?

When facing contradictory data about TMEM237 function across different experimental systems:

Systematic comparison of experimental conditions:

Resolution strategies for contradictory findings:

• Perform direct side-by-side comparisons:

  • Use identical protocols across different cell systems

  • Control for expression levels in overexpression studies

  • Apply consistent analytical methods for phenotype assessment

• Consider context-dependent functions:

  • TMEM237 may have different roles depending on:

    • Cell cycle stage

    • Ciliation state

    • Tissue microenvironment

    • Presence of specific interacting partners

• Examine isoform differences:

  • Determine if different splice variants are expressed in different systems

  • Verify which isoforms are detected by specific antibodies

  • Test isoform-specific functions through rescue experiments

• Integrate multi-level evidence:

  • Combine results from biochemical, cellular, and in vivo approaches

  • Weigh evidence based on experimental rigor and reproducibility

  • Consider evolutionary conservation of functions across species

• Develop unified models:

  • Propose models that can accommodate seemingly contradictory observations

  • Test predictions of these models with new experiments

  • Consider that TMEM237 may have pleiotropic functions (ciliary vs. non-ciliary)

This structured approach helps reconcile apparently contradictory findings and develops a more complete understanding of TMEM237 biology.

What are emerging applications of TMEM237 antibodies in studying ciliopathy mechanisms?

Emerging applications of TMEM237 antibodies in ciliopathy research include:

Single-cell analysis of transition zone composition:

• Using TMEM237 antibodies for high-resolution imaging of ciliary transition zone architecture

• Combining with super-resolution techniques to map precise spatial organization of transition zone proteins

• Correlating structural variations with ciliopathy phenotype severity

• Developing quantitative measurements of transition zone integrity

Proteome-wide interaction mapping:

• Employing TMEM237 antibodies for proximity labeling approaches (BioID, APEX)

• Identifying dynamic protein interactions at the transition zone under different conditions

• Creating comprehensive interaction networks of transition zone components

• Understanding how disease mutations alter these interaction networks

Patient-derived organoid studies:

• Using TMEM237 antibodies to characterize ciliary defects in patient-derived organoids

• Correlating TMEM237 expression and localization with tissue-specific phenotypes

• Testing pharmacological interventions to restore proper localization

• Developing personalized models for ciliopathy research

In vivo developmental studies:

• Applying TMEM237 antibodies in developmental studies of ciliopathy models

• Tracking tissue-specific expression patterns during embryogenesis

• Correlating TMEM237 dysfunction with organ-specific manifestations of Joubert syndrome

• Examining potential non-ciliary functions of TMEM237 during development

Therapeutic development and monitoring:

• Using TMEM237 antibodies to screen for compounds that restore proper protein localization

• Developing assays to monitor transition zone integrity in response to treatments

• Evaluating whether TMEM237 correction improves associated ciliary phenotypes

• Creating diagnostic tools for ciliopathy subtype identification

These emerging applications leverage TMEM237 antibodies to advance our understanding of transition zone biology and ciliopathy pathogenesis.

How might TMEM237 antibodies contribute to understanding the role of TMEM237 in cancer progression and potential therapeutic development?

TMEM237 antibodies offer several avenues for advancing cancer research and therapeutic development:

Diagnostic and prognostic applications:

• Developing IHC-based assays to assess TMEM237 expression in tumor biopsies

• Correlating expression levels with patient outcomes and treatment responses

• Creating standardized scoring systems for TMEM237 positivity in different cancer types

• Inclusion in multi-marker panels for cancer subtyping

Mechanistic research:

• Characterizing TMEM237-dependent signaling networks in cancer cells

• Identifying cancer-specific interaction partners through IP-MS approaches

• Mapping post-translational modifications that regulate TMEM237 function in tumors

• Investigating connections between hypoxia response and TMEM237 activation

Therapeutic target validation:

• Establishing in vivo models with TMEM237 modulation for drug testing

• Evaluating the effects of TMEM237 inhibition on tumor growth, metastasis, and EMT

• Screening for compounds that disrupt specific TMEM237 interactions (e.g., with NPHP1)

• Developing combination strategies targeting TMEM237-activated pathways

Monitoring treatment response:

• Using TMEM237 antibodies to track changes in expression/localization during treatment

• Correlating pathway inhibition (e.g., Pyk2/ERK) with TMEM237 complex formation

• Evaluating resistance mechanisms involving TMEM237 upregulation

• Developing liquid biopsy approaches to detect TMEM237-expressing circulating tumor cells

Translational research directions:

These approaches could ultimately lead to novel cancer diagnostics and therapeutics targeting TMEM237 or its downstream pathways.