ACVR1C Antibody

What are the key properties of ACVR1C antibodies?

ACVR1C antibodies are available in both polyclonal and monoclonal formats with different properties:

• Polyclonal antibodies: Typically raised in rabbits, recognize multiple epitopes on ACVR1C, demonstrating good sensitivity but potentially lower specificity

• Monoclonal antibodies: Often used for blocking experiments and therapeutic applications, recognizing single epitopes with higher specificity

• Molecular weight detection: ACVR1C typically appears at 55-68 kDa on Western blots

• Common applications: Western blot, ELISA, immunohistochemistry, and immunofluorescence

What species reactivity is available for commercial ACVR1C antibodies?

Most commercial ACVR1C antibodies demonstrate cross-reactivity with multiple species due to high sequence conservation:

The high conservation of ACVR1C across species, particularly in intracellular domains, enables broad cross-reactivity, especially for antibodies targeting conserved regions .

What are the recommended storage and handling protocols for ACVR1C antibodies?

For optimal preservation of ACVR1C antibody activity:

• Store at -20°C for long-term stability (up to one year)

• May be stored at 4°C for short-term use (up to three months)

• Most antibodies are supplied in PBS containing 0.02% sodium azide and 50% glycerol as stabilizers

• Avoid repeated freeze-thaw cycles which can degrade antibody quality

• For some small-volume antibodies (e.g., 20μL), aliquoting may be unnecessary for -20°C storage

• Working dilutions should be prepared fresh and kept cold during experiments

What controls should be included when using ACVR1C antibodies?

Proper experimental controls are essential for validating ACVR1C antibody results:

• Positive tissue controls: Human placenta lysate, mouse testis tissue, human pancreas tissue, and human brain tissue are recommended positive controls

• Negative controls: Samples lacking ACVR1C expression or tissues from knockout models

• Peptide competition: Some antibodies can be validated using specific blocking peptides (e.g., PA5-20597 can be used with blocking peptide PEP-0717)

• Isotype controls: Particularly important for flow cytometry and in vivo applications

• Knockout validation: Testing in ACVR1C knockout tissues provides definitive specificity confirmation

How can ACVR1C antibodies be optimized for detecting low-abundance expression in tissues?

Detecting low-abundance ACVR1C expression requires specific optimization strategies:

• Antigen retrieval optimization: Test both TE buffer (pH 9.0) and citrate buffer (pH 6.0) for optimal epitope exposure in FFPE tissues

• Signal amplification systems: Implement tyramide signal amplification (TSA) or polymer-based detection systems for IHC applications

• Extended antibody incubation: Optimize with overnight incubation at 4°C with carefully titrated antibody concentrations

• Background reduction: Implement thorough blocking steps to improve signal-to-noise ratio

• Application-specific dilutions: Start with recommended dilutions (WB: 1:1000-1:4000; IHC: 1:50-1:500) and optimize for specific tissue types

• Enhanced imaging: Utilize high-resolution or confocal microscopy for immunofluorescence applications

What are effective strategies for using ACVR1C antibodies in multiplex immunostaining?

When performing multiplex staining with ACVR1C antibodies:

• Select antibodies raised in different host species to enable specific secondary detection

• Consider directly conjugated primary antibodies to avoid cross-reactivity

• Implement sequential staining protocols for challenging combinations

• Validate that ACVR1C antibody performance remains consistent in multiplex contexts

• Address tissue autofluorescence through appropriate quenching methods

• Balance signal amplification to accommodate different expression levels

• Develop robust image analysis workflows for accurate co-localization quantification

• Include appropriate single-stain controls to establish accurate thresholds

How can researchers resolve contradictory results when using different ACVR1C antibodies?

When faced with inconsistent results from different ACVR1C antibodies:

• Epitope mapping: Determine which regions of ACVR1C are recognized by each antibody (e.g., N-terminal region between amino acids 130-180)

• Validation in knockout systems: Test all antibodies in systems where ACVR1C expression is genetically eliminated

• Recombinant protein controls: Use recombinant ACVR1C protein to standardize antibody responses

• Peptide competition: Perform blocking experiments with immunizing peptides to confirm specificity

• Cross-validation with orthogonal methods: Confirm antibody results with RT-PCR or RNA-seq

• Application-specific optimization: Some antibodies may perform better in certain applications

• Independent validation: Confirm key findings using antibodies from different suppliers targeting different epitopes

How can ACVR1C antibodies be used to study adipose tissue metabolism?

ACVR1C antibodies provide valuable tools for investigating adipose tissue metabolism:

• Neutralizing antibody studies: Anti-ACVR1C neutralizing antibodies administered in vivo (10 mg/kg s.c., weekly) can block ACVR1C signaling to study effects on adipose tissue metabolism

• Mechanistic pathway analysis: Combine ACVR1C antibodies with phospho-SMAD2/3 detection to trace activin signaling cascades that regulate adipogenesis and lipolysis

• Expression profiling: Characterize ACVR1C expression patterns across different adipose depots using immunohistochemistry to correlate with fat distribution phenotypes

• Temporal expression studies: Monitor ACVR1C expression during adipocyte differentiation, as it functions as "a novel marker specifically expressed during the late phase of adipocyte differentiation"

Research using these approaches has revealed that ACVR1C signaling suppresses adipose lipid mobilization, with inhibition leading to increased lipolysis, reduced fat mass, elevated plasma NEFAs and ketones, and potential metabolic complications including hepatic steatosis and insulin resistance .

What phenotypes are observed in studies using ACVR1C neutralizing antibodies in metabolic models?

Studies using anti-ACVR1C neutralizing antibodies in high-fat diet (HFD) models have demonstrated:

• Trend toward reduced body weight in HFD-fed mice

• Significant reduction in epididymal white adipose tissue weight

• Increased glycerol release and upregulation of lipolytic genes in adipose tissue

• Elevated plasma non-esterified fatty acids (NEFAs) and ketones

• Increased liver mass, hepatic fat accumulation, and elevated ALT levels

• Rapid increase in insulin levels, potentially indicating insulin resistance

These phenotypes closely mirror those observed in Inhbe-/- mice (lacking activin E), supporting a functional relationship between activin E and ACVR1C signaling in metabolic regulation .

How can ACVR1C antibodies be utilized in cancer immunotherapy research?

ACVR1C antibodies have emerging applications in cancer immunotherapy research:

• Flow cytometric analysis: Characterize ACVR1C expression across immune cell populations, particularly regulatory T cells (Tregs) where it is "uniquely expressed and highly upregulated during iTreg differentiation"

• Tumor models: Treat tumor-bearing mice with anti-ACVR1C antibodies to assess effects on cancer progression, as "mice deficient in Acvr1c were more resistant to cancer progression compared to wild type mice"

• Combination therapy studies: Test ACVR1C antibodies with checkpoint inhibitors, as "anti-tumor therapeutic effect was more significant when anti-Acvr1c antibody was administrated in combination with anti-PD-1 antibody"

• Mechanistic investigations: Combine ACVR1C and Foxp3 staining to track impacts on regulatory T cell differentiation and function

What is the role of ACVR1C in tumor microenvironment modulation?

ACVR1C antibody research has revealed important insights into tumor microenvironment regulation:

• Tumor-bearing mice and cancer patients show elevated levels of Activin A, which correlates with tumor burden

• Activin A promotes differentiation of conventional CD4+ T cells into immunosuppressive Foxp3+ induced Tregs, especially when TGF-β is limited

• ACVR1C is selectively expressed on regulatory T cells, making it a promising target for cancer immunotherapy

• Blocking ACVR1C with antibodies reduces Foxp3 expression in CD4+ T cells and enhances anti-tumor immunity

• Combined blockade of ACVR1C and PD-1 shows synergistic anti-tumor effects

These findings suggest that "blocking Activin A signaling through its receptor 1c is a promising and disease-specific strategy for preventing the accumulation of immunosuppressive iTregs in cancer" .

How should ACVR1C antibody results be interpreted in relation to SMAD2/3 signaling?

When studying ACVR1C in SMAD2/3 signaling contexts:

• ACVR1C functions as a type I receptor that, upon activation, phosphorylates SMAD2/3 transcription factors

• Interpret ACVR1C antibody results in relation to activin ligand specificity (activin AB, activin B, NODAL)

• Consider cell type-specific responses, as signaling outcomes vary between tissues (e.g., ACVR1C/SMAD2 signaling promotes invasion in retinoblastoma but modulates metabolism in adipocytes)

• For interaction studies, validate findings from tagged proteins with endogenous protein interactions

• Account for the temporal relationship between receptor blockade and changes in SMAD2/3 phosphorylation

• In retinoblastoma, ACVR1C levels are induced in invasive cases, while negative regulators like DACT2 and LEFTY2 are downregulated

What methodological approaches can detect ACVR1C-mediated SMAD activation?

To effectively study ACVR1C-mediated SMAD signaling:

• Use Western blot with phospho-specific SMAD2/3 antibodies to quantify pathway activation after ligand stimulation

• Implement co-immunoprecipitation with ACVR1C antibodies followed by SMAD2/3 detection to demonstrate physical interaction

• For transfection studies, use tagged constructs (such as "Flag-tagged Smad3 and HA-tagged ALK7 expression plasmids")

• Evaluate downstream transcriptional targets using ChIP assays with SMAD2/3 antibodies after ACVR1C activation or inhibition

• Perform comparative analysis between wild-type cells and those expressing ACVR1C variants (I195T, N150H, I482V) to assess signaling differences

• Monitor nuclear translocation of SMAD2/3 after ACVR1C activation using subcellular fractionation or immunofluorescence

What considerations are critical when using ACVR1C antibodies for in vivo blocking experiments?

When designing in vivo experiments with ACVR1C antibodies:

• Dosing regimen: Effective protocols typically use 10 mg/kg administered subcutaneously once weekly

• Duration limitations: Be aware that development of anti-human antibodies may limit studies beyond 4 weeks when using humanized antibodies in mice

• Control antibodies: Include appropriate isotype controls at equivalent concentrations

• Validation of blockade: Confirm effective ACVR1C blocking by assessing downstream signaling in collected tissues

• Combination effects: Consider potential interactions when combining with other treatments

• Genetic validation: Compare antibody blocking effects with genetic approaches (Acvr1c-/- mice) to confirm specificity

• Tissue collection timing: Plan tissue collection timing to coincide with optimal antibody activity period

How are therapeutic ACVR1C antibodies developed and characterized?

Development of therapeutic ACVR1C antibodies involves several key steps:

• Immunization strategy: Mice can be immunized with the extracellular domain (ECD) of ACVR1C to generate antibody-producing B lymphocytes

• Hybridoma technology: Fusion of antibody-producing B cells with immortal myeloma cells creates hybridomas producing monoclonal antibodies

• Clonal selection: Individual hybridoma clones are selected and expanded based on binding specificity and affinity

• In vivo production: "Hybridomas are injected into pristane-primed nude mice (2.5×10^6 cells/ml, 2ml per mouse) to generate ascites"

• Purification: Antibodies are purified from ascites fluid using protein G affinity chromatography

• Functional validation: Test antibodies for blocking capacity in cell-based assays measuring SMAD2/3 phosphorylation

• Epitope mapping: Determine binding sites through competition assays or peptide arrays

• Therapeutic assessment: Evaluate efficacy in disease models (metabolic disorders, cancer)

How can ACVR1C antibodies be used to characterize protein variants?

ACVR1C variants have been identified that correlate with altered body composition and metabolism:

• Exome-wide association studies identified variants (N150H, I195T, I482V) in ACVR1C associated with changes in waist-to-hip ratio

• When studying these variants, ACVR1C antibodies can:

  • Confirm equivalent protein expression levels of variants compared to wild-type receptors

  • Assess subcellular localization of each variant using immunofluorescence

  • Evaluate interaction with SMAD proteins through co-immunoprecipitation

  • Determine functional differences in signaling capacity by phospho-SMAD immunoblotting

  • Analyze receptor internalization and turnover rates

What genetic models are available for ACVR1C research?

Several genetic models have been developed for ACVR1C research:

• Global knockout mice: Generated using VelociGene technology to ablate the entire Acvr1c locus

• Conditional knockout models: Alk7fx/fx mice crossed with EIIa-cre mice to generate tissue-specific knockouts

• Point mutation knockin models: Mice carrying specific variants (I195T, N150H, I482V) generated on C57BL/6J background

• Expression validation: Transcript levels in these models can be verified by qPCR, while protein expression is confirmed using ACVR1C antibodies

These genetic models, in conjunction with appropriately validated ACVR1C antibodies, provide powerful tools for understanding the physiological roles of this receptor in metabolism, cancer biology, and development.