ARMC8 Antibody

What is ARMC8 and what are its key cellular functions?

ARMC8 (Armadillo Repeat-Containing Protein 8) is a conserved eukaryotic protein characterized by armadillo repeat domains. It plays important roles in regulating cell migration, proliferation, tissue maintenance, signal transduction, and tumorigenesis . ARMC8 functions as part of the CTLH E3 ubiquitin-protein ligase complex that mediates ubiquitination and subsequent proteasomal degradation of target proteins . Specifically, it mediates the ubiquitination and degradation of the transcription factor HBP1 .

ARMC8 is particularly important in regulating cell membrane adhesion complexes by promoting α-catenin degradation, which has significant implications for cell-cell adhesion and epithelial-mesenchymal transition (EMT) . Research has demonstrated that ARMC8 is overexpressed in several cancer types, including osteosarcoma, colon cancer, and non-small cell lung cancer, suggesting its role as a potential oncogene .

What are the known isoforms of ARMC8 and how do they differ structurally?

There are at least two documented isoforms of ARMC8: Armc8α and Armc8β. The first 364 amino acids of these isoforms are identical, but Armc8β has an early stop codon and encodes a shorter protein of 385 amino acids .

Armc8α possesses two distinct armadillo domains: a first domain comprising four armadillo repeats and a second domain with five armadillo repeats. These domains are connected by a large insert or loop region of approximately 150 amino acids . In contrast, Armc8β lacks the second armadillo domain entirely, which likely confers different functional properties . The full-length Armc8α protein contains 673 amino acids with a molecular weight of approximately 75.5 kDa .

What signaling pathways is ARMC8 known to interact with or regulate?

ARMC8 is primarily involved in the Wnt/β-catenin signaling pathway. Research has shown that knockdown of ARMC8 significantly inhibits the expression of β-catenin, c-Myc, and cyclin D1 in osteosarcoma cells, indicating that ARMC8 positively regulates this pathway .

Additionally, ARMC8 interacts with specific proteins in desmosomal complexes and adherens junctions, including specific δ-catenins (plakophilins-1, -2, -3 and p0071) and αE-catenin . It does not interact with αN-catenin or αT-catenin, suggesting specificity in its protein-protein interactions .

The TGF-β pathway has also been implicated in ARMC8 signaling networks, as indicated in pathway mapping studies . Through its multiple interactions with adhesion and signaling proteins, ARMC8 creates a complex regulatory network that influences cell behavior, particularly in the context of cancer progression and metastasis .

What are the recommended applications and optimal conditions for using ARMC8 antibodies in Western blot analysis?

For Western blot applications using ARMC8 antibodies, researchers should consider the following optimized conditions:

When performing Western blot analysis, it's important to note that ARMC8 antibodies can detect both endogenous expression in cancer cell lines and overexpressed recombinant protein . Some antibodies have been validated in transfected 293T cell lines showing a band at approximately 43 kDa for recombinant ARMC8 , which differs from the endogenous full-length protein (75.5 kDa).

For optimal results, researchers should include appropriate positive and negative controls, and may need to optimize blocking conditions and incubation times based on their specific experimental setup .

What protocols are recommended for immunohistochemistry and immunofluorescence applications with ARMC8 antibodies?

For immunohistochemistry (IHC) and immunofluorescence (IF) applications with ARMC8 antibodies:

For immunofluorescence, cells should be fixed (typically with 4% paraformaldehyde), permeabilized, and blocked with appropriate buffers containing gelatin or serum . Primary antibody incubation is typically performed for 2 hours at room temperature or overnight at 4°C, followed by washing steps and incubation with appropriate secondary antibodies .

Images can be acquired using confocal microscopy for high-resolution co-localization studies or standard fluorescence microscopy for general expression analysis . Both subcellular localization and expression levels of ARMC8 can be evaluated using these techniques.

How can researchers design and implement ARMC8 knockdown experiments to study its function?

Based on published research, effective ARMC8 knockdown experiments can be designed using the following approach:

• Selection of RNA interference method:

  • shRNA targeting ARMC8 mRNA (as demonstrated in osteosarcoma studies)

  • siRNA may also be effective for transient knockdown

• Verification of knockdown efficiency:

  • qRT-PCR to measure ARMC8 mRNA levels

  • Western blot to confirm protein reduction

  • Include appropriate controls (scrambled shRNA/siRNA)

• Cell line selection:

  • MG-63 and U2OS osteosarcoma cells have been successfully used

  • Other cancer cell lines with high ARMC8 expression may be suitable

• Functional assays to assess knockdown effects:

  • Cell proliferation assays (e.g., CCK-8 assay)

  • Migration assays (Transwell)

  • Invasion assays (Matrigel-coated membranes)

  • EMT marker expression analysis (E-cadherin, N-cadherin)

  • Western blot analysis for downstream signaling molecules (β-catenin, c-Myc, cyclin D1)

• In vivo validation:

  • Xenograft tumor models using cells with stable ARMC8 knockdown

  • Measurement of tumor volume and weight to assess growth inhibition

Research has demonstrated that ARMC8 knockdown significantly inhibits osteosarcoma cell proliferation in vitro and markedly reduces xenograft tumor growth in vivo . Additionally, ARMC8 silencing suppresses the EMT phenotype and inhibits migration and invasion of osteosarcoma cells, suggesting its importance in cancer progression .

How is ARMC8 expression linked to cancer progression and what methodologies are used to study this connection?

ARMC8 overexpression has been linked to multiple cancer types, and researchers use various methodologies to investigate this connection:

• Expression analysis methodologies:

  • qRT-PCR to measure ARMC8 mRNA levels in cancer tissues compared to normal tissues

  • Western blot to assess protein levels in cell lines and tissues

  • Immunohistochemistry to visualize expression patterns in tissue sections

• Clinical correlation approaches:

  • Analysis of ARMC8 expression in relation to TNM stage, lymph node metastasis, and patient prognosis

  • Comparison between tumor tissues and adjacent normal tissues

  • Correlation with other clinical parameters and biomarkers

• Functional studies:

  • Knockdown experiments using shRNA or siRNA to assess effects on cancer cell phenotypes

  • Cell proliferation assays (e.g., CCK-8)

  • Migration and invasion assays to assess metastatic potential

  • EMT marker analysis (E-cadherin, N-cadherin)

• Mechanistic investigations:

  • Analysis of Wnt/β-catenin pathway components (β-catenin, c-Myc, cyclin D1)

  • Assessment of cell adhesion complex integrity

Research has shown that ARMC8 is overexpressed in osteosarcoma cell lines compared to normal cells . Similar upregulation has been reported in colon cancer tissues compared to adjacent normal tissues, where ARMC8 expression was associated with TNM stage, lymph node metastasis, and poor prognosis . In non-small cell lung cancer, overexpression of ARMC8α was shown to promote growth, colony formation, and invasion in cancer cells .

What methodologies are used to study ARMC8 interactions with cell adhesion complexes?

To study ARMC8 interactions with cell adhesion complexes, researchers employ multiple complementary approaches:

• Yeast two-hybrid (Y2H) screening:

  • Gateway cloning of ARMC8 constructs into appropriate Y2H vectors

  • Testing interactions with specific proteins (e.g., δ-catenins, plakophilins, α-catenin)

  • Allows for systematic screening of potential interaction partners

• Co-immunoprecipitation techniques:

  • Immunoprecipitating ARMC8 and detecting interacting partners by Western blot

  • Using antibodies against potential partners to co-precipitate ARMC8

  • Can be performed in various cell types to confirm physiological relevance

• Co-localization studies:

  • Immunofluorescence microscopy to visualize spatial relationships between ARMC8 and adhesion proteins

  • Confocal microscopy for higher resolution co-localization analysis

  • Quantitative analysis of co-localization coefficients

• Domain mapping experiments:

  • Creating truncated constructs (e.g., C-terminal Armc8α, Armc8α 2nd Arm domain)

  • Testing which domains are required for specific protein interactions

  • Mutational analysis of key residues in interaction interfaces

Research has revealed that ARMC8 interacts specifically with certain δ-catenins (plakophilins-1, -2, -3 and p0071) and with αE-catenin, but not with αN-catenin or αT-catenin . These specific interactions suggest a role for ARMC8 in regulating desmosomal complexes and adherens junctions, potentially through mediating protein degradation .

How is ARMC8's role in epithelial-mesenchymal transition (EMT) experimentally assessed?

Researchers assess ARMC8's role in epithelial-mesenchymal transition (EMT) using multiple experimental approaches:

• EMT marker analysis:

  • Western blot analysis of epithelial markers (E-cadherin) and mesenchymal markers (N-cadherin)

  • Immunofluorescence to visualize changes in marker expression and localization

  • qRT-PCR to measure mRNA levels of EMT-related genes

• ARMC8 manipulation experiments:

  • Knockdown of ARMC8 using shRNA or siRNA to observe effects on EMT markers

  • Rescue experiments to confirm specificity of observed effects

• Functional assays for EMT phenotypes:

  • Migration assays (Transwell) to assess cell motility changes

  • Invasion assays (Matrigel-based) to evaluate invasive potential

  • Morphological assessment of cellular changes characteristic of EMT

• Signaling pathway analysis:

  • Western blot analysis of Wnt/β-catenin pathway components (β-catenin, c-Myc, cyclin D1)

  • Assessment of other EMT-related signaling pathways

Research has demonstrated that ARMC8 silencing in osteosarcoma cells induces an increase in E-cadherin expression paralleled by a decrease in N-cadherin expression, indicating suppression of the EMT phenotype . This alteration in EMT markers corresponds with reduced migration and invasion capabilities in ARMC8-silenced cells . These findings suggest that ARMC8 normally promotes EMT, consistent with its reported role in enhancing migration and invasion in cancer cells.

What approaches should researchers use to validate ARMC8 antibody specificity and troubleshoot non-specific binding?

To validate ARMC8 antibody specificity and address non-specific binding issues, researchers should employ a multi-faceted approach:

• Comprehensive antibody validation strategies:

  • Verify antibody specificity using ARMC8 knockdown or knockout systems

  • Test multiple antibodies targeting different epitopes of ARMC8

  • Compare results with published data and expected molecular weight (75.5 kDa)

  • Include positive controls (A549, HeLa, HepG2 cell lysates)

• Optimization of experimental conditions:

  • Titrate antibody concentration (starting with manufacturer's recommendations)

  • Test different blocking agents (BSA, non-fat milk, normal serum)

  • Optimize incubation times and temperatures

  • For IHC applications, compare different antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

• Technical considerations for Western blotting:

  • Use fresh samples with protease inhibitors

  • Optimize protein loading amount

  • Consider gradient gels for better separation

  • Increase washing steps to reduce background

  • Use highly specific secondary antibodies

• Advanced verification techniques:

  • Peptide competition assays to confirm epitope specificity

  • Mass spectrometry validation of immunoprecipitated proteins

  • Correlation of protein detection with mRNA expression data

When interpreting results, researchers should be aware that ARMC8 exists in multiple isoforms (Armc8α and Armc8β), which may appear as distinct bands on Western blots . Additionally, some antibodies detect recombinant ARMC8 at approximately 43 kDa, which differs from the endogenous full-length protein (75.5 kDa) .

How should researchers interpret conflicting data regarding ARMC8 function across different experimental systems?

When facing conflicting data regarding ARMC8 function across different experimental systems, researchers should consider several factors for proper interpretation:

• Context-dependent biological differences:

  • Different cell types and tissues may exhibit variable ARMC8 functions

  • Cancer versus normal cells may show distinct ARMC8 activities

  • The function of ARMC8 may depend on the expression of specific interaction partners

• Methodological considerations:

  • Different knockdown/knockout techniques may have varying efficiencies and off-target effects

  • Transient versus stable manipulation of ARMC8 may yield different results

  • Antibody specificity issues may lead to detection of different ARMC8 isoforms or cross-reactive proteins

• Analytical framework for resolving discrepancies:

  • Compare experimental conditions in detail (cell types, reagents, time points)

  • Evaluate the sensitivity and specificity of readout assays

  • Consider that ARMC8 functions through multiple mechanisms (protein degradation, signaling regulation)

  • Assess whether differences reflect isoform-specific functions (Armc8α versus Armc8β)

• Validation approaches:

  • Repeat experiments using multiple independent methods

  • Use rescue experiments with wild-type and mutant ARMC8 constructs

  • Perform dose-response studies to identify threshold effects

  • Combine in vitro and in vivo approaches for more comprehensive understanding

Research has shown that ARMC8 can function in both protein degradation pathways (as part of the CTLH E3 ubiquitin-protein ligase complex) and in signaling regulation (Wnt/β-catenin pathway) . These dual functions may contribute to apparently conflicting observations in different experimental systems.

What controls should be included when studying ARMC8 protein-protein interactions and degradation targets?

When investigating ARMC8 protein-protein interactions and degradation targets, researchers should implement a comprehensive set of controls:

• Essential controls for protein-protein interaction studies:

  • Input controls to verify protein expression levels

  • Negative controls using unrelated proteins or IgG for immunoprecipitation

  • Reciprocal co-immunoprecipitation to confirm interactions

  • Domain mapping controls using truncated constructs to identify interaction regions

  • Competition assays to verify binding specificity

• Controls for degradation mechanism studies:

  • Proteasome inhibitors (e.g., MG132) to confirm proteasome-dependent degradation

  • Cycloheximide chase assays to differentiate between degradation and synthesis effects

  • Ubiquitination assays with wild-type and mutant ubiquitin constructs

  • Controls with non-degradable substrate variants

• System-specific validation controls:

  • Use of multiple cell types to ensure generalizability

  • in vitro reconstitution experiments to confirm direct effects

  • Comparison of endogenous versus overexpressed proteins

  • Time-course experiments to establish degradation kinetics

• Pathway validation controls:

  • Manipulation of upstream and downstream pathway components

  • Pathway inhibitors to confirm mechanism (e.g., Wnt/β-catenin inhibitors)

  • Reporter assays to measure functional consequences of interactions

Research has established that ARMC8 promotes α-catenin degradation affecting cell membrane adhesion complexes and is part of the CTLH E3 ubiquitin-protein ligase complex that mediates ubiquitination and degradation of the transcription factor HBP1 . These findings highlight the importance of including appropriate controls to distinguish between direct and indirect effects of ARMC8 on protein degradation.

What experimental strategies can elucidate the dual role of ARMC8 in both proteasomal degradation and signaling pathway regulation?

To investigate ARMC8's dual functions in protein degradation and signaling regulation, researchers can implement several sophisticated experimental strategies:

• Structure-function dissection approaches:

  • Generate domain-specific mutants that separate degradation and signaling functions

  • Create chimeric proteins with domains from related proteins

  • Perform alanine scanning mutagenesis of key residues

  • Use CRISPR-Cas9 to introduce endogenous mutations in specific domains

• Temporal analysis methodologies:

  • Employ time-course experiments with high temporal resolution

  • Use inducible expression/knockdown systems (Tet-On/Off)

  • Apply optogenetic tools for precise temporal control of ARMC8 function

  • Perform pulse-chase experiments to track protein dynamics

• Advanced biochemical separation techniques:

  • Use size exclusion chromatography to isolate distinct ARMC8-containing complexes

  • Apply immunoaffinity purification with complex-specific antibodies

  • Implement BioID or APEX2 proximity labeling to identify context-specific interactors

  • Employ mass spectrometry to characterize complex composition

• Integrated pathway analysis:

  • Combine inhibitors of proteasomal degradation with pathway modulators

  • Use pathway-specific reporters (e.g., TOPFlash for Wnt/β-catenin)

  • Perform global phosphoproteomic analysis after ARMC8 manipulation

  • Apply systems biology approaches to model ARMC8 regulatory networks

Research indicates that ARMC8 functions as part of the CTLH E3 ubiquitin-protein ligase complex that mediates protein degradation while also regulating the Wnt/β-catenin signaling pathway . In osteosarcoma cells, ARMC8 silencing inhibits β-catenin, c-Myc, and cyclin D1 expression , suggesting that ARMC8 may coordinate protein degradation and signaling activation in a context-dependent manner.

How can researchers design experiments to identify and validate novel ARMC8 substrates in the ubiquitin-proteasome system?

To identify and validate novel ARMC8 substrates in the ubiquitin-proteasome system, researchers can implement a multi-dimensional experimental approach:

• Global proteomic screening methods:

  • Stable Isotope Labeling with Amino acids in Cell culture (SILAC) comparing control and ARMC8-depleted cells

  • Tandem Mass Tag (TMT) proteomics to quantify protein abundance changes

  • Pulse-SILAC to measure protein turnover rates

  • Filter candidates based on increased stability in ARMC8-depleted conditions

• Ubiquitinome analysis techniques:

  • Ubiquitin remnant profiling using K-ε-GG antibodies

  • Serial enrichment strategies for ubiquitinated proteins

  • Quantitative comparison of ubiquitination patterns

  • Ubiquitin chain topology analysis to characterize linkage types

• Direct biochemical validation approaches:

  • In vitro reconstitution of the CTLH E3 ligase complex with purified components

  • Cell-free degradation assays with candidate substrates

  • Ubiquitination assays with recombinant E1, E2, and the ARMC8-containing E3 complex

  • Mass spectrometry to identify ubiquitinated residues

• Cellular validation strategies:

  • Co-immunoprecipitation to confirm physical interaction with ARMC8

  • Cycloheximide chase assays to measure protein half-life

  • Proteasome inhibition to confirm degradation mechanism

  • Creation of lysine-mutant substrates resistant to ubiquitination

Research has established that ARMC8 is part of the CTLH E3 ubiquitin-protein ligase complex that mediates ubiquitination and subsequent proteasomal degradation of the transcription factor HBP1 . Additionally, ARMC8 promotes α-catenin degradation , suggesting it may target multiple substrates in different cellular contexts. These known interactions provide valuable positive controls for validation experiments.

What methodological approaches can examine the evolutionary conservation of ARMC8 function across species?

To study the evolutionary conservation of ARMC8 function across species, researchers can employ several sophisticated methodological approaches:

• Comparative genomic and phylogenetic analysis techniques:

  • Construct phylogenetic trees using ARMC8 sequences from diverse species

  • Perform codon-based analyses to identify selection pressures

  • Use synteny analysis to examine genomic context conservation

  • Identify conserved regulatory elements in promoter regions

• Cross-species functional complementation strategies:

  • Express ARMC8 orthologs from different species in human cell lines with ARMC8 knockdown

  • Assess rescue of phenotypes (proliferation, migration, EMT)

  • Compare biochemical properties of orthologs (interaction partners, degradation targets)

  • Create domain-swapped chimeric proteins between distant orthologs

• Model organism experimental approaches:

  • Generate knockout/knockdown models in evolutionarily diverse organisms

  • Compare phenotypes across species

  • Assess conservation of molecular mechanisms (e.g., Wnt/β-catenin regulation)

  • Examine tissue-specific functions in different model systems

• Molecular interaction conservation studies:

  • Compare ARMC8 interaction partners across species using affinity purification-mass spectrometry

  • Test conservation of specific interactions (e.g., with plakophilins, α-catenin)

  • Perform cross-species yeast two-hybrid screens

  • Map conservation of post-translational modifications

Research has established that ARMC8 is an evolutionarily conserved armadillo protein involved in cell-cell adhesion complexes through multiple molecular interactions . The armadillo repeat structure, which is critical for protein-protein interactions, is maintained across diverse species, suggesting functional conservation of ARMC8's role in regulating cell adhesion and signaling pathways throughout evolution.

What emerging research areas are likely to advance our understanding of ARMC8 function in normal and disease states?

Several emerging research areas have significant potential to advance our understanding of ARMC8 biology:

• Single-cell analysis approaches:

  • Single-cell transcriptomics to identify cell populations with differential ARMC8 expression

  • Spatial transcriptomics to map ARMC8 expression in tissue contexts

  • Single-cell proteomics to correlate ARMC8 with protein networks at individual cell level

  • These approaches could reveal cell type-specific functions of ARMC8 not apparent in bulk analysis

• Structural biology and protein engineering:

  • Cryo-EM structures of ARMC8 within the CTLH E3 ligase complex

  • Structure-guided development of selective inhibitors

  • Engineered ARMC8 variants with enhanced or altered specificity

  • These studies could enable precise modulation of specific ARMC8 functions

• Integrated multi-omics:

  • Combining transcriptomics, proteomics, and metabolomics after ARMC8 manipulation

  • Network analysis to identify key nodes in ARMC8-regulated pathways

  • Correlation of genomic alterations with ARMC8 function in patient samples

  • These integrative approaches could reveal broader impacts of ARMC8 on cellular homeostasis

• Therapeutic targeting strategies:

  • Development of proteolysis-targeting chimeras (PROTACs) targeting ARMC8

  • Small molecule inhibitors of ARMC8 protein interactions

  • Peptide inhibitors that disrupt specific ARMC8 functions

  • These approaches could translate ARMC8 biology into potential cancer therapeutics

Research has established ARMC8's importance in cancer progression through effects on proliferation, invasion, and EMT . As a component of the CTLH E3 ubiquitin-protein ligase complex and regulator of cell adhesion complexes , ARMC8 represents a promising target for therapeutic development, particularly in cancers where it is overexpressed.

What methodological challenges remain in the field of ARMC8 research and potential strategies to overcome them?

Despite significant progress, several methodological challenges persist in ARMC8 research:

• Isoform-specific analysis limitations:

  • Challenge: Distinguishing functions of ARMC8 isoforms (Armc8α and Armc8β)

  • Solution strategies:

    • Develop isoform-specific antibodies with validated specificity

    • Use CRISPR-Cas9 to selectively target individual isoforms

    • Create isoform-specific knockin reporter systems

• Complex formation and dynamics:

  • Challenge: Understanding the dynamics of ARMC8 incorporation into different complexes

  • Solution strategies:

    • Implement live-cell imaging with fluorescently tagged ARMC8

    • Use FRET/BRET techniques to monitor protein-protein interactions in real-time

    • Apply super-resolution microscopy to visualize complex formation

• Substrate identification limitations:

  • Challenge: Comprehensive identification of physiological ARMC8 substrates

  • Solution strategies:

    • Combine multiple proteomics approaches (SILAC, ubiquitinome analysis)

    • Develop engineered ARMC8 variants that trap substrates

    • Implement proximity labeling in different cellular compartments

• In vivo functional analysis:

  • Challenge: Understanding ARMC8 function in complex in vivo environments

  • Solution strategies:

    • Generate conditional tissue-specific knockout models

    • Develop in vivo imaging methods to track ARMC8 activity

    • Implement patient-derived xenograft models to study cancer relevance

The complexity of ARMC8 function, involving both protein degradation mechanisms as part of the CTLH E3 ligase complex and signaling regulation through the Wnt/β-catenin pathway , presents significant challenges. Overcoming these limitations will require interdisciplinary approaches combining advanced molecular biology techniques with systems-level analysis.