TMEM209 Antibody

What is TMEM209 and where is it normally expressed?

TMEM209 (Transmembrane Protein 209) is an integral nuclear envelope protein that, under normal physiological conditions, exhibits highly tissue-specific expression. Northern blot analysis has identified a 3.5-kb transcript of TMEM209 specifically in the testis, with minimal to no detectable expression in other healthy human tissues examined . This restricted expression pattern makes TMEM209 particularly interesting as a potential cancer biomarker and therapeutic target, as its expression in other tissues may indicate pathological processes. The protein was initially identified as an integral component of the nuclear envelope in mouse liver through high-throughput shotgun proteomics using multidimensional protein identification technology .

What are the available TMEM209 antibodies and their binding specificities?

Several TMEM209 antibodies are commercially available, with different binding specificities and applications. Common variants include:

• N-Terminal binding antibodies: These recognize epitopes at the N-terminus of TMEM209 protein. Examples include polyclonal antibodies generated using synthetic peptides from the N-terminus of human TMEM209 (Q96SK2, NP_116231) .

• Mid-region binding antibodies: Some antibodies target the amino acid region 251-519 of the TMEM209 protein, offering alternative epitope recognition .

The choice between these antibodies depends on the experimental goals, as different epitopes may be more or less accessible depending on protein conformation, interactions, and cellular localization.

What species cross-reactivity can be expected with TMEM209 antibodies?

TMEM209 antibodies show variable cross-reactivity across species due to sequence conservation. Based on BLAST analysis, the following reactivity profiles can be expected:

• High conservation (100% identity): Human, Chimpanzee, Gorilla, Gibbon, Monkey, Galago, Marmoset, Elephant, Bat, Horse, Pig, Opossum, Turkey, Chicken, Platypus

• Moderate conservation (92-93% identity): Mouse, Panda, Dog, Bovine, Rabbit, Guinea pig

• Lower conservation (85% identity): Rat

When selecting an antibody for cross-species applications, researchers should consider these homology percentages to anticipate potential reactivity. It is advisable to validate the antibody in each new species before proceeding with full experiments, even when high sequence identity suggests cross-reactivity should occur.

What is the subcellular localization of TMEM209 protein?

Immunocytochemical analysis of lung cancer SBC-5 cells that overexpress endogenous TMEM209 has revealed that the protein localizes to multiple subcellular compartments. Specifically, TMEM209 is detected:

• On the nuclear envelope (primary location)

• In the Golgi apparatus

• Weakly in the cytoplasm

This multicompartmental distribution suggests TMEM209 may have distinct functions depending on its cellular location. The predominant localization at the nuclear envelope aligns with its identified interaction with nuclear pore complex components, particularly NUP205, which influences nuclear transport processes.

How does TMEM209 contribute to cancer cell growth and survival mechanisms?

TMEM209 has been identified as a critical driver of human lung cancer growth and survival through several mechanisms:

• Nuclear pore complex interaction: TMEM209 interacts with nucleoporin protein NUP205, stabilizing it and consequently increasing the level of c-Myc in the nucleus . This interaction appears to be crucial for cancer cell proliferation.

• Cell cycle regulation: Flow cytometric analysis has demonstrated that siRNA-mediated knockdown of TMEM209 results in G1 arrest in SBC-5 lung cancer cells, indicating its role in cell cycle progression .

• Selective growth advantage: Overexpression of TMEM209 promotes cancer cell growth, while its attenuation through siRNA is sufficient to block growth in lung cancer cell lines that highly express this protein .

The cancer-specific expression pattern of TMEM209 (normally limited to testis but widely expressed in lung cancer) makes it particularly interesting as a therapeutic target, as inhibiting its function could potentially affect cancer cells while sparing most normal tissues.

What methods are most effective for studying TMEM209-protein interactions?

Based on published research methodologies, the following approaches have proven effective for studying TMEM209 protein interactions:

• Coimmunoprecipitation followed by mass spectrometry: This approach successfully identified the interaction between TMEM209 and NUP205. The methodology involves:

  • Transfection of cells with TMEM209 expression vector or mock vector

  • Preclearing of cell extracts with protein G-agarose beads

  • Immunoprecipitation using anti-Flag M2 agarose

  • Multiple washing steps with immunoprecipitation buffer

  • Protein separation using gradient SDS-PAGE gel

  • Silver staining for protein visualization

  • Excision of specific protein bands for matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis

• Functional validation through RNA interference: siRNA-mediated knockdown of TMEM209 followed by assessment of interacting partner levels and localization can confirm the functional significance of identified interactions .

• Subcellular co-localization studies: Immunofluorescence microscopy to determine whether TMEM209 and its interacting partners co-localize in specific cellular compartments, providing spatial context for the interactions.

These methodological approaches can be applied to identify and characterize novel TMEM209-interacting proteins beyond NUP205.

What are the optimal conditions for Western blotting using TMEM209 antibodies?

For optimal Western blotting results with TMEM209 antibodies, the following conditions are recommended based on experimental protocols:

• Antibody dilution: Using a concentration of 0.2-1 μg/mL in 5% skim milk/PBS buffer provides optimal signal-to-noise ratio .

• Secondary antibody: HRP-conjugated anti-Rabbit IgG should be diluted at 1:50,000-1:100,000 for optimal detection .

• Sample preparation: Since TMEM209 is a membrane-associated protein found in the nuclear envelope and Golgi apparatus, effective membrane protein extraction methods should be employed, potentially including detergents like NP-40 (0.5%) as used in immunoprecipitation buffers .

• Expected molecular weight: TMEM209 appears at approximately 63 kDa on Western blots , which should be considered when evaluating band specificity.

• Controls: For validation, using cell lysates from cells with known TMEM209 expression is recommended. Lung cancer cell lines such as SBC-5 and LC319 show high endogenous expression, while SBC-3 cells show minimal expression and can serve as a negative control .

It's important to note that optimal working dilutions should be determined experimentally by each investigator, as conditions may vary depending on sample type and detection system .

How can researchers validate the specificity of TMEM209 knockdown effects in functional studies?

When performing TMEM209 knockdown studies, ensuring specificity of the observed effects is critical. The following validation approaches are recommended:

• Multiple siRNA sequences: Use at least two different siRNA sequences targeting TMEM209 (such as si-TMEM209-#1 and si-TMEM209-#2 as described in the literature) to confirm that the observed phenotype is consistent across different targeting sequences .

• Appropriate controls: Include both non-targeting control siRNAs (such as si-EGFP or si-LUC) to control for non-specific effects of the siRNA transfection process .

• Rescue experiments: Perform rescue experiments by expressing an siRNA-resistant version of TMEM209 to determine if the phenotype can be reversed.

• Cell line selectivity: Test TMEM209 siRNAs in cell lines with different levels of endogenous TMEM209 expression. For example, research has shown that while TMEM209 siRNAs significantly reduced viability in LC319 and SBC-5 cells (which highly express TMEM209), they had no effect on SBC-3 cells with minimal TMEM209 expression, indicating specificity rather than off-target effects .

• Phenotype characterization: Thoroughly characterize the phenotypic effects of TMEM209 knockdown using multiple assays, such as:

  • Western blotting to confirm protein reduction

  • Colony formation assays

  • MTT viability assays

  • Flow cytometry for cell cycle analysis

This multi-faceted approach helps ensure that the observed cellular effects are specifically due to TMEM209 depletion rather than off-target effects.

What is the relationship between TMEM209 and the nuclear pore complex in cancer progression?

The relationship between TMEM209 and the nuclear pore complex (NPC) appears to be central to its role in cancer progression:

• Direct interaction with NUP205: Mass spectrometric analysis has identified the nucleoporin protein NUP205 as a TMEM209-interacting protein. This interaction appears to stabilize NUP205 .

• Regulation of nuclear transport: Through its interaction with NUP205 and integration into nuclear envelope structures, TMEM209 likely influences selective nuclear transport of proteins critical for cancer progression.

• Modulation of c-Myc levels: TMEM209-NUP205 interaction increases the level of c-Myc in the nucleus . c-Myc is a well-established oncogenic transcription factor that regulates cell proliferation, metabolism, and survival.

• Potential therapeutic target: The TMEM209-NUP205 interaction represents a specific molecular interaction that could be targeted therapeutically. Disrupting this interaction could potentially inhibit cancer cell growth while having minimal effects on normal cells given the cancer-specific expression pattern of TMEM209 .

Researchers studying this relationship should consider approaches to specifically disrupt the TMEM209-NUP205 interaction, such as developing peptide inhibitors or small molecules that could interfere with the binding interface between these proteins.

How should researchers design experiments to study TMEM209 expression in clinical samples?

When designing experiments to study TMEM209 expression in clinical samples, researchers should consider the following approach based on successful published methodologies:

• Sample selection and cohort design:

  • Include diverse tumor types and subtypes (previous studies examined 120 lung cancer cases including different histological types)

  • Include matched normal adjacent tissue when possible

  • Consider including tissue microarrays for higher throughput screening

• Multi-level expression analysis:

  • mRNA expression: RT-PCR using validated primers for TMEM209 can detect 3-fold or higher expression in tumor samples compared to normal tissues

  • Protein expression: Western blotting with validated TMEM209 antibodies

  • Tissue localization: Immunohistochemistry to examine subcellular distribution patterns

• Data validation strategies:

  • Use multiple detection methods (e.g., RT-PCR, Western blot, immunohistochemistry)

  • Compare results with existing datasets or literature

  • Include positive controls (testis tissue, which normally expresses TMEM209) and negative controls (normal tissues that typically lack TMEM209 expression)

• Clinical correlation analysis:

  • Correlate TMEM209 expression with clinicopathological parameters (stage, grade, subtype)

  • Perform survival analysis to determine prognostic significance

  • Consider multivariate analysis to assess independence of TMEM209 as a marker

This comprehensive approach allows for robust characterization of TMEM209 expression patterns in clinical samples and evaluation of its potential as a biomarker or therapeutic target.

What are the considerations for developing targeted therapies against TMEM209?

Developing targeted therapies against TMEM209 requires careful consideration of several factors:

• Target validation:

  • Confirm the oncogenic role of TMEM209 through knockdown and overexpression studies in multiple cancer models

  • Evaluate the effects of TMEM209 inhibition on normal cells to predict potential toxicities

  • Determine whether TMEM209's function is essential in all cancer types or specific to certain malignancies

• Target accessibility:

  • Consider TMEM209's subcellular localization (nuclear envelope, Golgi, cytoplasm)

  • Evaluate which domains or regions of the protein are accessible to potential therapeutic agents

  • Determine whether targeting the protein directly or its interactions (e.g., with NUP205) would be more effective

• Therapeutic approaches:

  • Small molecule inhibitors targeting protein-protein interactions (particularly TMEM209-NUP205)

  • Antisense oligonucleotides or siRNA to reduce TMEM209 expression

  • Antibody-drug conjugates targeting cancer-specific epitopes of TMEM209

  • PROTAC (Proteolysis Targeting Chimera) approaches to induce degradation of TMEM209

• Specificity considerations:

  • Leverage the cancer-specific expression pattern of TMEM209 (normal expression limited to testis)

  • Design therapeutics that maximize the therapeutic window between cancer and normal cells

  • Consider potential effects on germline cells given testicular expression

• Combination approaches:

  • Investigate synergistic effects of TMEM209 inhibition with standard-of-care therapies

  • Explore combinations with other targeted therapies, particularly those affecting nuclear transport or c-Myc pathways

These considerations highlight both the potential and challenges of developing TMEM209-targeted therapies, with the cancer-specific expression pattern offering a promising foundation for selective cancer treatment.

How can researchers effectively use TMEM209 antibodies for immunofluorescence studies?

For effective immunofluorescence studies using TMEM209 antibodies, researchers should consider the following methodological recommendations:

• Fixation and permeabilization optimization:

  • Since TMEM209 localizes to multiple cellular compartments including the nuclear envelope and Golgi apparatus , a combination of paraformaldehyde fixation (3-4%) followed by permeabilization with Triton X-100 (0.1-0.5%) typically provides good results

  • For detailed nuclear envelope visualization, additional permeabilization steps may be necessary to ensure antibody access to this compartment

• Antibody selection and dilution:

  • Use antibodies validated for immunofluorescence applications

  • Start with manufacturer-recommended dilutions (typically 1:100 to 1:500) and optimize as needed

  • Consider using N-terminal targeting antibodies as these regions may be more accessible in fixed cells

• Co-staining strategies:

  • Combine TMEM209 staining with markers for specific cellular compartments to confirm localization:

    • Nuclear envelope: Lamin B1 or nuclear pore complex proteins like NUP205

    • Golgi apparatus: GM130 or other Golgi markers

    • For cancer studies, include markers related to c-Myc signaling or cell proliferation

• Controls and validation:

  • Positive controls: Use cell lines known to express TMEM209 (e.g., SBC-5, LC319)

  • Negative controls: Include cell lines with minimal TMEM209 expression (e.g., SBC-3) or cells treated with TMEM209 siRNA

  • Peptide competition assays can confirm antibody specificity

• Advanced imaging techniques:

  • Super-resolution microscopy can provide detailed localization information, particularly useful for nuclear envelope studies

  • Live-cell imaging using fluorescently tagged TMEM209 can complement fixed-cell studies and provide dynamic information

Following these guidelines will help researchers obtain reliable and informative immunofluorescence data on TMEM209 localization and its changes in different experimental conditions or disease states.

What are the key unanswered questions about TMEM209 function in cancer biology?

Despite significant progress in understanding TMEM209's role in cancer, several important questions remain unanswered:

• Mechanistic understanding: While the interaction with NUP205 and effects on c-Myc nuclear levels have been established , the precise molecular mechanisms by which TMEM209 influences nuclear transport selectivity remain to be fully elucidated.

• Cancer specificity: Why is TMEM209 specifically upregulated in certain cancers despite its normal restriction to testis tissue? The transcriptional and epigenetic mechanisms controlling this cancer-specific expression pattern warrant further investigation.

• Broader oncogenic significance: Current studies have focused primarily on lung cancer . Whether TMEM209 plays similar roles in other cancer types and whether its expression correlates with specific oncogenic mutations or subtypes remains to be systematically explored.

• Therapeutic targeting potential: The feasibility of developing specific inhibitors of TMEM209 or its protein interactions, and whether such inhibitors would show selective toxicity toward cancer cells, represents a critical area for further research.

• Biomarker value: The potential utility of TMEM209 as a diagnostic, prognostic, or predictive biomarker across different cancer types needs more comprehensive evaluation with larger patient cohorts.