CMTM2 Antibody

What is CMTM2 and what biological roles does current research indicate it plays?

CMTM2 belongs to the CMTM family that forms a bridge between chemokines and the transmembrane 4 superfamily. This protein is highly expressed in testis, bone marrow, and peripheral blood cells, indicating its diverse physiological roles . Research has established CMTM2's involvement in multiple biological processes including hematopoiesis, spermatogenesis, and membrane apposition . More significantly, CMTM2 has been identified as a potential antioncogene in several cancer types including embryonal carcinoma, yolk sac tumor, and non-small cell lung cancer . Recent investigations have also revealed CMTM2's role in viral pathogenesis, with studies showing it can negatively control HIV-1 transcription by targeting the AP-1 and CREB pathways in T-cells .

What are the key technical specifications researchers should know when selecting CMTM2 antibodies?

When selecting CMTM2 antibodies for research, investigators should consider several critical specifications that determine experimental compatibility and reliability:

Researchers should note that commercially available antibodies like the CMTM2 Polyclonal Antibody have been verified with specific cell lines such as K562, which should be considered when planning experiments requiring positive controls .

Why does CMTM2 frequently show unexpected molecular weights in Western blot analysis?

The discrepancy between calculated (27 kDa) and observed molecular weights of CMTM2 in Western blot analysis stems from several biological and technical factors. Western blotting detects proteins in complex samples based on specific antigen-antibody binding after mobility-based separation . Multiple factors affect protein mobility, potentially causing band sizes to differ from theoretical predictions . Most notably, post-translational modifications can generate multiple bands simultaneously when a protein exists in different modified states within the same sample . For membrane proteins like CMTM2, this variance is particularly common due to glycosylation, phosphorylation, or other modifications that alter electrophoretic mobility. Researchers should anticipate these discrepancies and employ appropriate positive controls to confirm band identity.

What is the optimal protocol for CMTM2 detection via Western blotting?

A robust Western blotting protocol for CMTM2 detection requires careful consideration of the protein's membrane-bound nature. Begin sample preparation by lysing cells on ice using a lysis buffer supplemented with 1% protease inhibitor and 0.5% PMSF to prevent degradation . Determine protein concentrations using BCA assays and prepare samples in SDS electrophoresis buffer with thorough heat denaturation at 100°C . After electrophoretic separation and membrane transfer, block with skimmed milk and incubate membranes with primary anti-CMTM2 antibody at 4°C for 24 hours at the recommended dilution (1:1000-1:5000) . Following incubation with appropriate HRP-conjugated secondary antibody, visualize immunoblots using ECL reagents and analyze using densitometry software . When interpreting results, remember that the observed molecular weight may differ from the calculated 27 kDa due to post-translational modifications . Including verified positive controls such as K562 cell lysates enhances result reliability .

How can researchers quantitatively measure CMTM2 levels in serum or culture supernatants?

ELISA represents the gold standard for quantitative measurement of CMTM2 in liquid samples such as serum or culture supernatants. When implementing this methodology, researchers should follow manufacturer-recommended protocols while ensuring proper sample handling to preserve protein integrity . The general workflow involves sample collection under standardized conditions, followed by precise adherence to the ELISA kit protocol which typically includes plate coating, blocking, sample addition, antibody incubation, washing steps, substrate development, and optical density measurement . For optimal results, researchers should generate standard curves using purified recombinant CMTM2 protein and ensure samples fall within the linear range of detection. This approach has been successfully employed to demonstrate significantly lower serum CMTM2 concentrations in patients with HBV-related liver diseases compared to healthy individuals .

What experimental approach should be used to investigate CMTM2 ubiquitination and degradation mechanisms?

To investigate CMTM2 ubiquitination and degradation mechanisms, researchers should employ a comprehensive ubiquitination assay protocol. Begin by transfecting cells (e.g., HepG2) with the expression vector of interest (such as HBx) or appropriate control vectors . Treat a subset of transfected cells with proteasome inhibitors (30 μM MG132 for 24 hours) to block protein degradation . After treatment, lyse cells in RIPA buffer supplemented with 1% phenylmethylsulfonyl fluoride and 5% N-ethylmaleimide to preserve ubiquitination . Immunoprecipitate CMTM2 by incubating the lysate with anti-CMTM2 antibody overnight at 4°C, followed by protein A/G addition and further incubation . Analyze both total protein and immunoprecipitated samples via Western blotting using antibodies against ubiquitin, K48-linked ubiquitin, or K63-linked ubiquitin to assess ubiquitination patterns . This approach has revealed that HBx-activated K48-linked polyubiquitination drives CMTM2 degradation, which can be blocked by proteasome inhibition .

How does serum CMTM2 correlate with clinical parameters in HBV-related liver diseases?

Analysis of serum CMTM2 levels reveals distinct correlation patterns with clinical parameters across different HBV-related liver diseases:

What evidence supports CMTM2 as a potential biomarker for HBV-related disorders?

Multiple lines of evidence support CMTM2's potential as a biomarker for HBV-related disorders. First, ELISA analyses demonstrate significantly lower serum CMTM2 concentrations in patients with CHB, HBLC, and HCC compared to healthy controls (p <0.001) . Second, receiver operating characteristic (ROC) curve analysis indicates significant association between CMTM2 levels and the diagnostic value of HBV-related disorders . Third, the negative correlation between serum CMTM2 and HBV DNA levels in CHB patients provides a measurable relationship with viral replication . Fourth, experimental evidence from cellular models confirms that HBV infection directly suppresses CMTM2 expression through HBx-activated ubiquitin-proteasome degradation, establishing a mechanistic basis for its biomarker potential . The consistency across clinical samples and cellular models strengthens CMTM2's candidacy as a biomarker, though additional large-scale validation studies would be required before clinical implementation.

How does CMTM2 influence cancer cell behavior in experimental models?

Experimental manipulation of CMTM2 expression reveals its significant impact on cancer cell behaviors, particularly in hepatocellular carcinoma models:

In HepG2 cells, CMTM2 knockdown significantly enhances proliferation as measured by CCK-8 assays, while overexpression inhibits growth . Flow cytometry analysis reveals that these effects occur through cell cycle modulation rather than apoptosis induction . Specifically, CMTM2 appears to induce G2/M phase arrest, with knockdown reducing G2/M phase accumulation and overexpression increasing it . Additionally, transwell migration and invasion assays demonstrate that CMTM2 silencing significantly enhances cell motility and invasive capacity . Together, these findings suggest CMTM2 functions as a tumor suppressor in liver cancer models by restricting cell cycle progression and inhibiting metastatic behaviors.

What are common technical challenges when working with CMTM2 antibodies and how can they be addressed?

When working with CMTM2 antibodies, researchers frequently encounter several technical challenges that require specific troubleshooting approaches. First, the membrane localization of CMTM2 necessitates effective extraction methods using appropriate detergents to solubilize membrane proteins completely . Second, the discrepancy between calculated (27 kDa) and observed molecular weights requires careful band interpretation and validation using positive controls . Third, since CMTM2 belongs to a family of related proteins, antibody cross-reactivity must be assessed through specificity controls including knockdown experiments or peptide competition assays. Fourth, low endogenous expression levels in certain cell types may necessitate signal enhancement techniques such as extended exposure times or more sensitive detection systems. To address these challenges, researchers should optimize sample preparation protocols specifically for membrane proteins, validate antibody specificity using multiple approaches, include appropriate positive controls in each experiment, and consider application-specific optimizations such as extended incubation times or enhanced detection methods.

How should researchers address contradictory data when analyzing CMTM2 expression across different experimental systems?

When confronting contradictory CMTM2 expression data across different experimental systems, researchers should implement a systematic analytical approach. First, evaluate methodological differences that might explain discrepancies, including antibody clones used, detection methods, and sample preparation techniques. Second, consider biological variables such as cell type-specific expression patterns, growth conditions, and the presence of regulatory factors that might modulate CMTM2 levels. Third, examine temporal factors, as CMTM2 expression may vary dynamically during disease progression or cellular responses. Fourth, assess technical variables including protein extraction efficiency from membrane fractions, which can significantly impact detection. Fifth, when comparing in vitro and in vivo data, acknowledge the complexity of the in vivo microenvironment which includes multiple regulatory factors absent in cell culture. To resolve contradictions, researchers should perform controlled comparative studies using standardized protocols across systems, employ multiple detection methods simultaneously, and conduct time-course experiments to capture dynamic expression changes.

What controls are essential for validating CMTM2 antibody specificity in research applications?

Rigorous validation of CMTM2 antibody specificity requires implementation of multiple complementary controls. Primary validation should include expression manipulation controls where CMTM2 is overexpressed or knocked down/knocked out, with corresponding changes in signal intensity confirming antibody specificity . Peptide competition assays represent another critical control, where pre-incubation of the antibody with immunizing peptide should substantially reduce or eliminate specific signals. Cross-reactivity assessment using samples from different species or testing against related CMTM family members helps establish binding selectivity. Positive control samples with verified CMTM2 expression (such as K562 cells for Western blotting) should be included in each experiment . For immunoprecipitation applications, mass spectrometry confirmation of pulled-down proteins provides definitive validation. When feasible, employing multiple antibodies targeting different CMTM2 epitopes and observing consistent detection patterns offers compelling evidence of specificity. These comprehensive controls collectively minimize the risk of misinterpreting experimental results due to non-specific antibody reactions.

How does HBV infection regulate CMTM2 expression through the ubiquitin-proteasome system?

HBV infection suppresses CMTM2 expression through a specific ubiquitin-proteasome pathway orchestrated by the HBV X protein (HBx). Experimental evidence demonstrates that CMTM2 protein levels are significantly decreased in HBV-expressing cellular models, including both HepG2.2.15 cells (which constitutively produce HBV) and HepG2 cells transfected with HBV genome constructs (pcDNA-HBV1.3 and pcDNA-HBV1.1) . Mechanistically, HBx activates Lys48 (K48)-linked polyubiquitination of CMTM2, specifically targeting it for proteasomal degradation . This degradation process is effectively blocked by treatment with the proteasome inhibitor MG132, confirming the essential role of the proteasome pathway . The specificity of K48-linked ubiquitination (rather than K63-linked) suggests a precise degradation mechanism rather than other ubiquitin-mediated cellular processes . This HBV-driven suppression of CMTM2 may contribute to viral persistence and disease progression by neutralizing CMTM2's potential antiviral or tumor-suppressive functions, representing a novel mechanism by which HBV manipulates host cellular pathways.

What molecular pathways connect CMTM2 function to cell cycle regulation and cancer progression?

The molecular mechanisms through which CMTM2 regulates cell cycle progression and cancer development involve complex interactions with cell cycle machinery. Flow cytometry analysis demonstrates that CMTM2 specifically induces G2/M phase arrest without triggering apoptosis . Knockdown of CMTM2 reduces G2/M phase accumulation while promoting cell proliferation, migration, and invasion in HepG2 cells . Conversely, CMTM2 overexpression increases G2/M population while inhibiting proliferation . These observations suggest CMTM2 may interact with key G2/M checkpoint regulators such as cyclin B1/CDK1 complexes, Wee1 kinase, or CDC25 phosphatases, though specific interaction partners require further investigation. Beyond cell cycle effects, CMTM2's inhibition of migration and invasion suggests potential involvement in epithelial-mesenchymal transition pathways or regulation of matrix metalloproteinases. The tumor-suppressive properties align with clinical observations of reduced CMTM2 levels in cancers, including HBV-related HCC . Future research should focus on identifying direct CMTM2 interaction partners and mapping its position within cancer-relevant signaling networks using techniques such as proximity labeling, phosphoproteomics, and pathway analysis.

What are emerging hypotheses about CMTM2's role in immune regulation and virus-host interactions?

Emerging evidence suggests CMTM2 may function at the intersection of immune regulation and virus-host interactions, though many aspects remain hypothetical. CMTM2's classification within the chemokine-like factor superfamily implies potential immunomodulatory functions, particularly given its expression in bone marrow and peripheral blood cells . Its previously documented ability to negatively regulate HIV-1 transcription through AP-1 and CREB pathways in T-cells suggests a broader antiviral role that may extend to other viruses including HBV . The negative correlation between serum CMTM2 levels and HBV viral load in CHB patients further supports a potential antiviral function . HBV's evolutionary strategy to degrade CMTM2 through the ubiquitin-proteasome system indicates its importance in virus-host dynamics . Future research should explore whether CMTM2 influences innate immune signaling pathways such as interferon responses, pattern recognition receptor signaling, or inflammasome activation. Additionally, investigations into CMTM2's potential role in adaptive immunity, including effects on T-cell and B-cell functions, would further illuminate its position in immune regulation. Understanding these mechanisms could reveal new therapeutic targets for viral infections and associated malignancies.