TMEM120B Antibody

What is TMEM120B and what are its known biological functions?

TMEM120B (transmembrane protein 120B) is a 339 amino acid multi-pass membrane protein belonging to the TMEM120 family . It localizes to cellular membranes and has a calculated molecular weight of 40 kDa, though it typically appears at approximately 50 kDa in Western blot applications . While its precise function was previously unknown, recent research has revealed that TMEM120B plays significant roles in cancer biology.

It has been identified as a protein that interacts with myosin heavy chain 9 (MYH9) and activates the β1-integrin/FAK-TAZ-mTOR signaling axis, which maintains stemness and accelerates chemotherapy resistance in breast cancer cells . TMEM120B contains six transmembrane domains and a coil-coil domain and is located on chromosome 12q24.31 . Its homolog TMEM120A (also known as NET29) is involved in adipogenesis, but TMEM120B appears to have distinct functions in malignant tissues .

What are the specific characteristics of the TMEM120B antibody 24539-1-AP?

The TMEM120B antibody (24539-1-AP) is a rabbit polyclonal IgG antibody produced using a TMEM120B fusion protein (Ag21609) as the immunogen . The antibody has been validated for Western blot (WB) and ELISA applications, with recommended Western blot dilutions of 1:500-1:1000 . It shows specific reactivity with human and mouse samples, with positive detection confirmed in mouse cerebellum tissue .

The antibody is supplied as a liquid in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, with a concentration of approximately 850 μg/ml by Nanodrop and 540 μg/ml by Bradford method . It is purified through antigen affinity purification, which enhances its specificity for the target protein . The antibody recognizes the full protein with the observed molecular weight of 50 kDa, slightly higher than the calculated weight of 40 kDa, which may indicate post-translational modifications of the target protein .

What are the optimal conditions for using TMEM120B antibody in Western blot experiments?

For optimal Western blot results with the TMEM120B antibody (24539-1-AP), researchers should use the following methodology:

• Sample preparation: Fresh tissue samples or cultured cells should be lysed in standard RIPA buffer supplemented with protease inhibitors. Mouse cerebellum tissue has been validated as a positive control for this antibody .

• Protein loading and separation: Load 20-40 μg of total protein per lane on SDS-PAGE gels (10-12% acrylamide is recommended for proteins in the 50 kDa range).

• Transfer conditions: Use standard wet or semi-dry transfer protocols with PVDF or nitrocellulose membranes.

• Blocking: Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute the TMEM120B antibody at 1:500-1:1000 in blocking buffer and incubate at 4°C overnight or at room temperature for 1.5 hours .

• Secondary antibody: Use an appropriate anti-rabbit HRP-conjugated secondary antibody.

• Detection: Standard ECL reagents are suitable for detection of the signal.

It's important to note that the antibody should be titrated in each testing system to obtain optimal results, as the optimal conditions may be sample-dependent .

What sample types and preparation methods are most effective for TMEM120B antibody applications?

The following sample types and preparation methods have proven effective for TMEM120B antibody applications:

• Tissue samples: Mouse cerebellum tissue has been validated as a positive control for Western blot applications . For human tissue samples, fresh or frozen breast cancer tissues have shown reliable results in research studies .

• Cell lines: The antibody has been used successfully with various breast cancer cell lines including MCF-7, SK-BR-3, MDA-453, and MDA-231 . These cell lines serve as excellent models for studying TMEM120B expression and function.

• Sample preparation protocol:

  • For tissues: Homogenize tissues in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 8.0) containing protease inhibitors.

  • For cultured cells: Wash cells with cold PBS, then lyse directly in the plate using RIPA buffer with protease inhibitors. Incubate on ice for 15-30 minutes, then scrape and collect lysates.

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

  • Quantify protein concentrations using Bradford or BCA assay.

  • Prepare samples for SDS-PAGE by mixing with Laemmli buffer and heating at 95°C for 5 minutes.

For immunohistochemistry (IHC) applications shown in research studies, paraffin-embedded tissue sections were processed using standard antigen retrieval methods before antibody application .

How can TMEM120B antibody be used to investigate cancer progression and chemotherapy resistance?

TMEM120B antibody can be instrumental in investigating cancer progression and chemotherapy resistance through multiple methodological approaches:

• Expression profiling in cancer tissues: Researchers can use IHC staining with TMEM120B antibody to evaluate expression patterns across various cancer types. Studies have demonstrated that TMEM120B expression is elevated in lung, breast, gastric, colon, and ovarian cancers compared to normal tissues . The antibody can help researchers establish correlations between TMEM120B expression levels and clinicopathological factors such as TNM stage, lymph node metastasis, and patient prognosis .

• Mechanistic studies of chemotherapy resistance: By combining TMEM120B antibody with cellular models of drug resistance, researchers can:

  • Perform Western blot analysis to compare TMEM120B expression levels between chemosensitive and chemoresistant cell lines

  • Use the antibody in immunoprecipitation experiments to identify binding partners like MYH9

  • Conduct immunofluorescence studies to examine cellular localization changes during development of resistance

• Signaling pathway analysis: The antibody can be used to investigate the relationship between TMEM120B and the TAZ-mTOR signaling pathway, which has been implicated in stemness and chemotherapy resistance:

  • Co-immunoprecipitation experiments to confirm protein-protein interactions

  • Western blot analysis of phosphorylated pathway components after TMEM120B overexpression or knockout

  • Correlation analysis between TMEM120B and downstream effectors in patient samples

• Functional validation studies: Researchers can employ the antibody to confirm TMEM120B-specific effects in:

  • Sphere formation assays to assess cancer stemness properties

  • Colony formation and cell proliferation assays following manipulation of TMEM120B expression

  • Xenograft models to evaluate in vivo effects of TMEM120B on tumor growth and drug resistance

What is the role of TMEM120B in the β1-integrin/FAK-TAZ-mTOR signaling axis, and how can it be studied?

TMEM120B plays a critical role in the β1-integrin/FAK-TAZ-mTOR signaling axis, which is essential for maintaining cancer cell stemness and chemotherapy resistance. This role can be studied through several methodological approaches using the TMEM120B antibody:

• Protein interaction studies:

  • Co-immunoprecipitation (Co-IP): TMEM120B antibody can be used to pull down protein complexes, followed by Western blot detection of interaction partners such as MYH9 . This approach revealed that TMEM120B directly binds to the coil-coil domain of MYH9, which is crucial for focal adhesion assembly.

  • GST pull-down assays: These can complement Co-IP results to verify direct protein-protein interactions between TMEM120B and its binding partners .

  • Mass spectrometry analysis: After immunoprecipitation with TMEM120B antibody, LC-MS/MS can identify novel interaction partners as demonstrated in the research where TMEM120B-MYH9 interaction was characterized .

• Signaling pathway activation assessment:

  • Western blot analysis of pathway components: Following TMEM120B overexpression or knockdown, researchers can use specific antibodies to detect changes in phosphorylation status of FAK, TAZ, and mTOR.

  • Immunofluorescence microscopy: Co-staining of TMEM120B with focal adhesion markers (e.g., paxillin, vinculin) and TAZ can reveal spatial relationships and co-localization patterns.

• Functional domain analysis:

  • Comparison between wild-type TMEM120B and TMEM120B-ΔCCD (deletion of coil-coil domain): Studies showed that TMEM120B-ΔCCD delayed focal adhesion formation, suppressed TAZ-mTOR signaling, and abrogated chemotherapy resistance, highlighting the importance of the coil-coil domain .

• Protein stability and degradation studies:

  • Ubiquitination assays: Research revealed that TMEM120B stabilizes MYH9 by preventing its degradation by CUL9 in a ubiquitin-dependent manner .

  • Cycloheximide chase assays: These can determine protein half-life changes of MYH9 in the presence or absence of TMEM120B.

Methodologically, these studies require careful optimization of antibody concentrations, incubation conditions, and washing steps to ensure specific detection of protein interactions and pathway components.

What are common challenges when using TMEM120B antibody in experimental procedures, and how can they be addressed?

Researchers may encounter several challenges when working with TMEM120B antibody. Here are common issues and their solutions:

• Non-specific bands in Western blot:

  • Challenge: Multiple bands appearing at unexpected molecular weights.

  • Solution: Optimize antibody dilution (start with 1:500-1:1000 as recommended) . Include proper positive controls (mouse cerebellum tissue) . Increase washing stringency with TBST and extend blocking time to reduce background. Consider using gradient gels for better separation around the 50 kDa region where TMEM120B is detected.

• Weak or no signal detection:

  • Challenge: Inability to detect TMEM120B even in positive control samples.

  • Solution: Ensure adequate protein loading (40-60 μg for tissue samples). Verify transfer efficiency using reversible protein staining. Fresh preparation of the antibody dilution from stock may improve detection. Consider longer primary antibody incubation (overnight at 4°C) and optimize ECL exposure times.

• Inconsistent results across different sample types:

  • Challenge: Variable detection efficiency between different tissue or cell types.

  • Solution: The manufacturer notes that results may be sample-dependent . Adjust extraction methods based on the sample type, potentially using more stringent lysis buffers for membrane proteins. Consider using phosphatase inhibitors in addition to protease inhibitors during sample preparation.

• Discrepancy between calculated and observed molecular weight:

  • Challenge: TMEM120B appears at 50 kDa rather than the calculated 40 kDa .

  • Solution: This is an expected observation for TMEM120B and likely represents post-translational modifications. Researchers should anticipate detection at approximately 50 kDa. Using protein deglycosylation assays or phosphatase treatments prior to Western blot may help characterize these modifications.

• Cross-reactivity concerns:

  • Challenge: Potential cross-reactivity with TMEM120A, a homolog of TMEM120B.

  • Solution: Include appropriate control samples (TMEM120B knockout or knockdown) to confirm antibody specificity. Consider parallel detection with another TMEM120B antibody targeting a different epitope to validate results.

How should researchers interpret Western blot data for TMEM120B in relation to cancer progression markers?

When interpreting Western blot data for TMEM120B in cancer research, researchers should consider the following methodological guidelines:

• Expression level quantification:

  • Normalize TMEM120B expression to appropriate housekeeping proteins (β-actin, GAPDH, or tubulin).

  • Use densitometry software (ImageJ, Image Lab) to quantify relative expression levels.

  • Present data as fold change relative to normal tissue controls or appropriate cell line controls.

• Correlation with clinical parameters:

  • When analyzing patient samples, stratify TMEM120B expression data by TNM stage, lymph node metastasis status, and other relevant clinical parameters.

  • Research has shown that TMEM120B expression positively correlates with advanced TNM stage (p = 0.011) and lymph node metastasis (p = 0.006) .

  • Consider Kaplan-Meier survival analysis to correlate TMEM120B expression with patient outcomes, as studies have shown higher expression in patients with poor prognosis .

• Multi-marker analysis approach:

  • Analyze TMEM120B expression in conjunction with established cancer progression markers:

    • Stemness markers (SOX2, CD44, CD133)

    • TAZ and mTOR pathway components (phosphorylated mTOR, S6K)

    • Focal adhesion proteins (FAK, paxillin)

  • Establish correlation patterns using Spearman's correlation analysis between TMEM120B and these markers .

• Interpretation of mechanistic relationships:

  • Increases in TMEM120B should be evaluated alongside changes in MYH9 levels, as TMEM120B stabilizes MYH9 by preventing CUL9-mediated degradation .

  • Changes in focal adhesion assembly efficiency following TMEM120B manipulation provide functional validation of its role in cell motility and invasion.

  • Alterations in TAZ nuclear localization following TMEM120B overexpression or knockdown confirm its role in activating this signaling pathway.

• Data validation approaches:

  • Confirm Western blot findings using orthogonal techniques (qPCR, immunofluorescence).

  • Validate functional consequences through proliferation, invasion, and sphere formation assays.

  • Compare results across multiple cell lines to ensure reproducibility and biological relevance.

What are emerging research areas for TMEM120B in cancer biology beyond breast cancer?

Several emerging research areas for TMEM120B are being explored beyond breast cancer:

• Pan-cancer expression analysis:

  • Bioinformatic analyses from The Cancer Genome Atlas (TCGA) database have revealed that TMEM120B expression is elevated in multiple cancer types compared to normal tissues .

  • Immunohistochemical validation has confirmed high TMEM120B expression in lung, gastric, colon, and ovarian cancers .

  • Methodological approach: Researchers can use tissue microarrays with TMEM120B antibody to conduct comprehensive expression profiling across diverse cancer types, correlating expression with histopathological features and clinical outcomes.

• Role in metastasis and tumor microenvironment:

  • Given TMEM120B's involvement in focal adhesion assembly and cell invasion, its role in metastatic cascades warrants further investigation.

  • Methodological approach: Co-culture systems combining TMEM120B-manipulated cancer cells with stromal components can reveal how this protein influences tumor-stroma interactions. Live cell imaging with labeled focal adhesion components can visualize dynamic changes in cell motility following TMEM120B modulation.

• Therapeutic targeting strategies:

  • TMEM120B-MYH9 interaction represents a potential therapeutic target for overcoming chemotherapy resistance.

  • Methodological approach: Structure-function analysis using truncated TMEM120B constructs can identify critical interaction domains. High-throughput screening assays can be developed to identify small molecules that disrupt TMEM120B-MYH9 binding.

• Role in cancer stem cell regulation beyond TAZ signaling:

  • While TMEM120B's role in TAZ-mTOR activation has been established, its potential involvement in other stemness-related pathways remains unexplored.

  • Methodological approach: RNA-sequencing following TMEM120B modulation can identify additional affected pathways. ChIP-seq for stemness-related transcription factors can determine whether TMEM120B indirectly affects gene expression programs.

• Potential as a biomarker for treatment response:

  • TMEM120B expression was elevated in breast cancer patients with poor treatment outcomes (Miller/Payne grades 1-2) compared to those with better outcomes (Miller/Payne grades 3-5) .

  • Methodological approach: Prospective studies measuring TMEM120B expression in pre-treatment biopsies followed by correlation with treatment response can validate its utility as a predictive biomarker.

What novel methodological approaches are being developed for studying TMEM120B function?

Innovative methodological approaches are expanding our understanding of TMEM120B function:

• Advanced protein interaction mapping:

  • Proximity labeling techniques (BioID, APEX) coupled with mass spectrometry analysis provide spatial context for TMEM120B interactions in living cells.

  • Cross-linking mass spectrometry (XL-MS) can identify direct binding interfaces between TMEM120B and partners like MYH9.

  • Implementation: Fusion of TMEM120B with promiscuous biotin ligases allows for biotinylation of proximal proteins in their native cellular environment, followed by streptavidin pulldown and mass spectrometry identification.

• Live imaging of TMEM120B dynamics:

  • CRISPR-Cas9 genome editing to tag endogenous TMEM120B with fluorescent proteins enables real-time visualization of its localization and trafficking.

  • Fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) assays provide spatial and temporal information about TMEM120B-MYH9 interactions.

  • Implementation: Dual-color live imaging of fluorescently tagged TMEM120B and focal adhesion markers can reveal dynamic association during cell migration and invasion processes.

• Single-cell analysis approaches:

  • Single-cell RNA sequencing combined with TMEM120B expression profiling can reveal heterogeneity within tumor populations.

  • Mass cytometry (CyTOF) with TMEM120B antibodies allows simultaneous measurement of multiple proteins at the single-cell level.

  • Implementation: Correlation of TMEM120B levels with stemness markers at the single-cell level can identify specific subpopulations driving chemoresistance and tumor progression.

• Structural biology studies:

  • Cryo-electron microscopy of TMEM120B-containing complexes can elucidate the three-dimensional organization of interaction networks.

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map conformational changes upon binding partner interactions.

  • Implementation: Structural insights can guide the development of peptide inhibitors or small molecules targeting the TMEM120B-MYH9 interface.

• In vivo functional validation:

  • Conditional knockout mouse models specific for TMEM120B in mammary tissue can reveal its role in tumor initiation and progression.

  • Patient-derived xenograft (PDX) models with TMEM120B manipulation allow testing of its role in tumor growth and drug response in physiologically relevant conditions.

  • Implementation: Treatment of TMEM120B-overexpressing and control xenografts with docetaxel or doxorubicin can validate its role in chemotherapy resistance in vivo.