METTL7B Antibody

What is METTL7B and what cellular functions does it perform?

METTL7B (methyltransferase-like 7B) is a protein that functions as an alkyl thiol methyltransferase (TMT). It catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to hydrogen sulfide (H₂S) and other exogenous thiol small molecules. Research has confirmed that METTL7B can methylate several thiol compounds, including H₂S, 7α-thiospironolactone, L-penicillamine, and captopril, in a time- and concentration-dependent manner . METTL7B does not methylate endogenous thiols such as glutathione and cysteine, nor does it work on classic substrates for other known small molecule S-, N-, and O-methyltransferases . The protein has a molecular weight of approximately 28 kDa and contains a putative AdoMet-binding domain .

In which tissue types is METTL7B predominantly expressed?

METTL7B expression has been detected in various human, mouse, and rat tissues. In humans, METTL7B has been found to be overexpressed in several cancer types compared to normal tissues, particularly in lung adenocarcinoma (LUAD) and glioma . In rodent models, METTL7B has been detected in liver tissues, which is reflected in the typical positive Western blot controls used for METTL7B antibodies (mouse liver tissue, rat liver tissue) .

What are the recommended applications for METTL7B antibodies?

METTL7B antibodies have been validated for several experimental applications:

It is recommended to optimize dilution in each testing system to obtain optimal results, as this may be sample-dependent .

How should METTL7B antibodies be properly stored and handled to maintain reactivity?

Proper storage of METTL7B antibodies is critical for maintaining their reactivity. Most commercial METTL7B antibodies should be stored at -20°C and are typically stable for one year after shipment. The antibodies are generally provided in PBS buffer with 0.02% sodium azide and 50% glycerol at pH 7.3 . For long-term storage at -20°C, aliquoting is generally unnecessary, though smaller sized aliquots (e.g., 20μl) may contain 0.1% BSA as a stabilizer . When working with the antibody, avoid repeated freeze-thaw cycles which can degrade antibody quality and affect experimental reproducibility.

What is the optimal protocol for detecting METTL7B in paraffin-embedded tissue sections?

For immunohistochemistry detection of METTL7B in paraffin-embedded tissues, the following methodological approach is recommended:

• Prepare paraffin sections at 4-6μm thickness

• Perform antigen retrieval with TE buffer pH 9.0 (alternatively, citrate buffer pH 6.0 may be used)

• Block endogenous peroxidases with 3% hydrogen peroxide

• Apply the METTL7B antibody at a dilution of 1:50-1:500, optimized for your specific tissue

• Incubate overnight at 4°C

• Apply appropriate secondary antibody and develop using standard detection systems

This protocol has been validated particularly for mouse liver tissue, where METTL7B shows positive staining . When analyzing human cancer tissues, such as LUAD or glioma, METTL7B expression has been correlated with clinicopathological features including tumor size, TNM stages, and lymph node metastasis .

How can METTL7B expression be effectively modulated in cell lines for functional studies?

Several effective approaches have been documented for modulating METTL7B expression in cell culture models:

Knockdown methods:

• siRNA transfection: HepG2 cells treated with METTL7B-specific siRNA showed approximately 60% decreased METTL7B mRNA expression compared to scramble siRNA controls .

• shRNA lentiviral vectors: In A549 lung cancer cells, shRNA targeting METTL7B significantly reduced expression as verified by western blot .

Overexpression methods:

• Plasmid transfection: HeLa cells treated with a constitutive overexpression plasmid containing FLAG-tagged METTL7B showed more than 1000-fold increased mRNA expression compared to empty vector controls .

• Lentiviral vectors: H1299 lung cancer cells transfected with METTL7B-overexpressing lentiviral vectors (pcDNA-METTL7B) showed significantly increased METTL7B expression .

To verify successful modulation, both mRNA expression (via qRT-PCR) and protein expression (via western blot) should be assessed.

How is METTL7B expression correlated with cancer prognosis and what methodologies are used to assess this?

METTL7B expression has been significantly associated with cancer prognosis in multiple tumor types. The methodologies used to assess this correlation typically include:

The correlation between METTL7B expression and specific clinical features often appears as follows:

What methodologies are recommended for studying the relationship between METTL7B and immune infiltration in tumors?

Several comprehensive methodologies have been employed to study the relationship between METTL7B and immune infiltration:

• ESTIMATE algorithm: This computational method can be used to calculate immune scores, stromal scores, and ESTIMATE scores from RNA-seq data. Studies have shown significant differences in these scores between patients with high and low METTL7B expression levels .

• Cibersort algorithm: This deconvolution approach analyzes the proportions of 22 immune cell subsets from gene expression data. Research has revealed significant differences in T cells CD8, NK cells activated, Monocyte, Macrophages M1, Macrophages M2, and Neutrophils between groups with different METTL7B expression levels .

• Spearman correlation analysis: This statistical method can be used to assess correlations between METTL7B expression and specific immune cells. METTL7B has been found to correlate with multiple immune cells, including Neutrophils, Macrophages M1, Macrophages M2, and various T cell populations .

• TIMER database: This web resource allows exploration of the relationship between gene expression and immune cell infiltration. Analysis using TIMER has shown that METTL7B expression correlates with B cells, CD8+ T cells, CD4+ T cells, neutrophils, macrophages, and dendritic cells in various cancer types .

• Gene Set Enrichment Analysis (GSEA): This computational method identifies significantly enriched gene sets. GSEA has revealed that METTL7B is associated with multiple immune-related functions and pathways .

What are the technical considerations when using recombinant METTL7B for enzymatic activity assays?

When working with recombinant METTL7B for enzymatic activity assays, several technical considerations should be addressed:

• Expression system selection: LOBSTR-BL21(DE3) E. coli cells have been successfully used for METTL7B expression. Cells should be grown in ampicillin-containing terrific broth (TB) expression media at a ratio of 1:100 (overnight culture to fresh media) .

• Induction conditions: METTL7B production should be initiated via addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. For optimal protein folding, the temperature should be reduced to 15°C after induction, with cells grown for an additional 24 hours .

• Protein purification strategy: METTL7B can be expressed with affinity tags to aid in solubilization and purification. A fusion protein with two affinity tags (His-GST-METTL7B) has been successfully used. Cell pellets should be thawed and processed in a cold environment (4°C) .

• Enzyme activity assay design: For methylation activity assays, several substrates can be tested, including H₂S, 7α-thiospironolactone, L-penicillamine, and captopril. The methylation reaction should be monitored in a time- and concentration-dependent manner. Note that endogenous thiols such as glutathione and cysteine are not substrates for METTL7B .

• Controls: Include S-adenosyl-L-homocysteine (AdoHcy) as a competitive inhibitor control. Additionally, mutating the conserved aspartate residue at position 98 to alanine (D98A) can serve as a negative control, as this mutation abolishes methylation activity .

How can researchers address discrepancies between METTL7B expression patterns observed in different experimental systems?

When facing discrepancies in METTL7B expression patterns across different experimental systems, consider the following methodological approaches:

• Cell-specific expression variation: Studies have shown variable METTL7B expression across different cell lines. For example, A549 lung cancer cells show higher METTL7B expression compared to H1299 cells . Always establish baseline expression in your specific cell system through qRT-PCR and western blot before proceeding with functional studies.

• Tissue vs. cell line discrepancies: Some studies have reported contradictory findings between tissue samples and cell lines. For instance, while METTL7B is overexpressed in lung adenocarcinoma tissues compared to normal tissues, some studies found decreased expression in LUAD cell lines compared to normal lung epithelial cells (BEAS-2B) . These discrepancies might be due to:

  • Loss of in vivo microenvironmental factors in cell culture

  • Clonal selection during cell line establishment

  • Passage-dependent changes in gene expression

• Antibody-related considerations: Different antibodies may target different epitopes of METTL7B, leading to discrepant results. When western blotting, a band at ~58 kDa is visible using anti-METTL7A and anti-GST antibodies but not with anti-METTL7B antibodies . Always validate antibodies using positive and negative controls, and consider using multiple antibodies targeting different epitopes.

• Methodological variations: Differences in sample preparation, RNA extraction methods, and normalization strategies can contribute to discrepancies. Standardize protocols across experiments and incorporate appropriate technical and biological replicates.

• Data normalization approaches: For transcriptomic data analysis, batch effects can be corrected using packages like LIMMA and SVA . When integrating data from multiple sources, employ proper batch correction methods to minimize technical variation.

What are the most effective strategies for studying METTL7B's role in modulating immune responses in cancer models?

To effectively study METTL7B's role in modulating immune responses in cancer models, consider implementing these comprehensive strategies:

• In vitro co-culture systems: Establish co-culture systems using METTL7B-modulated cancer cells (via knockdown or overexpression) with immune cells such as T cells, B cells, or macrophages. This approach can help evaluate direct effects of METTL7B on immune cell recruitment, activation, and function.

• Flow cytometry analysis: Use multi-parameter flow cytometry to assess immune cell populations and their activation status in METTL7B-manipulated models. This technique allows for detailed characterization of immune cell subsets and their functional markers.

• Cytokine profiling: Measure cytokine and chemokine production using techniques such as ELISA, multiplex bead arrays, or cytokine profiling assays. Research suggests that METTL7B expression correlates with M2 chemokines more strongly than M1 chemokines, indicating a potential role in immunosuppressive microenvironments .

• Immune checkpoint analysis: Investigate the relationship between METTL7B and immune checkpoint molecules (PD-1, PD-L1, CTLA-4, LAG3, TIM3) using techniques such as qRT-PCR, western blot, or flow cytometry. Studies have shown positive correlations between METTL7B expression and multiple immune checkpoints .

• In vivo models with immune component analysis: Develop xenograft or syngeneic mouse models with METTL7B-modulated cancer cells and analyze tumor growth alongside immune infiltration. Use immunohistochemistry or mass cytometry to characterize the tumor immune microenvironment.

• Single-cell RNA sequencing: Apply single-cell transcriptomics to dissect the heterogeneity of immune populations in relation to METTL7B expression. This approach can reveal cell-specific effects and identify rare immune cell populations affected by METTL7B.

• Functional T cell assays: Implement T cell proliferation, cytotoxicity, and exhaustion assays to functionally characterize the impact of METTL7B on anti-tumor immunity. This is particularly relevant given the correlation between METTL7B and T cell populations in tumor samples .

These methodologies should be selected based on your specific research question and available resources, with appropriate controls included to validate findings.

What are the emerging approaches for targeting METTL7B in cancer therapeutics, and how can researchers evaluate their efficacy?

Several emerging approaches for targeting METTL7B in cancer therapeutics are being explored, with specific methodologies for evaluating their efficacy:

• Small molecule inhibitors: Designing specific inhibitors targeting METTL7B's methyltransferase activity can be a promising approach. Efficacy can be evaluated through:

  • Enzymatic assays using recombinant METTL7B and known substrates (captopril, H₂S)

  • Cell viability assays in cancer cell lines with high METTL7B expression

  • Comparison with genetic knockdown effects to confirm target specificity

• RNA interference strategies: Beyond laboratory siRNA/shRNA approaches, therapeutic RNA interference strategies could be developed. These can be evaluated through:

  • In vivo delivery efficiency using nanoparticles or lipid-based carriers

  • Sustained knockdown duration in animal models

  • Effects on tumor growth, metastasis, and immune infiltration

• Immunotherapy combinations: Given METTL7B's association with immune checkpoints and immune cell infiltration, combining METTL7B inhibition with immunotherapy may enhance efficacy. Evaluation approaches include:

  • Analysis of PD-1, PD-L1, CTLA-4 expression after METTL7B modulation

  • In vivo models assessing combinatorial effects with immune checkpoint inhibitors

  • Monitoring changes in tumor immune microenvironment composition

• Biomarker development: METTL7B expression could serve as a predictive biomarker for treatment response. Methods for evaluation include:

  • Correlation of METTL7B expression with treatment outcomes in patient cohorts

  • Development of standardized IHC or liquid biopsy assays for clinical use

  • Multivariate analysis incorporating METTL7B with other predictive factors

• Targeting downstream pathways: Research has implicated METTL7B in multiple pathways, including cell cycle regulation and various signaling pathways (TNF, NF-kappaB, MAPK) . Efficacy of targeting these downstream effects can be assessed through:

  • Phosphoproteomic analysis before and after METTL7B modulation

  • Cell cycle analysis using flow cytometry

  • Combination treatment strategies with established pathway inhibitors

How can researchers effectively study the interplay between METTL7B and hydrogen sulfide (H₂S) signaling in physiological and pathological conditions?

To effectively study the interplay between METTL7B and hydrogen sulfide (H₂S) signaling, researchers should consider these methodological approaches:

• H₂S detection techniques:

  • Use fluorescent probes specific for H₂S detection (such as SF7-AM or WSP-5)

  • Employ polarographic H₂S sensors for real-time measurements

  • Implement the methylene blue assay for quantitative H₂S determination

  • Consider mass spectrometry-based approaches for detecting methylated H₂S metabolites

• Genetic modulation studies:

  • Create METTL7B knockdown/knockout and overexpression models in relevant cell types

  • Simultaneously manipulate H₂S-producing enzymes (CBS, CSE, 3MST) to study combined effects

  • Use CRISPR-Cas9 to introduce specific mutations in the METTL7B active site (such as D98A) to disrupt methylation activity

• Enzyme activity assays:

  • Develop robust in vitro enzymatic assays to measure METTL7B-mediated methylation of H₂S

  • Implement kinetic analysis to determine enzyme parameters (Km, Vmax, kcat)

  • Study the effects of physiological and pathological factors on METTL7B activity

• Redox state analysis:

  • Assess changes in cellular redox state when modulating METTL7B expression

  • Measure reactive oxygen species (ROS) levels using fluorescent probes

  • Quantify glutathione levels and glutathione/glutathione disulfide ratios

  • Evaluate protein S-sulfhydration using modified biotin switch techniques

• Signaling pathway analysis:

  • Investigate how METTL7B-mediated H₂S methylation affects key signaling pathways

  • Use phosphoproteomic approaches to identify affected signaling nodes

  • Implement transcriptomic analysis to identify genes responsive to H₂S signaling modulation

  • Correlate findings with physiological outcomes (cell growth, migration, etc.)