ANKMY2 Antibody

What is ANKMY2 and what cellular functions does it perform?

ANKMY2 (Ankyrin repeat and MYND domain containing 2) is a 441 amino acid protein containing three ANK repeats and one MYND-type zinc finger domain. It is encoded by a gene mapping to human chromosome 7p21.1 and is highly conserved across multiple species including chimpanzee, dog, cow, mouse, chicken, zebrafish, fruit fly, mosquito, and Caenorhabditis elegans .

ANKMY2 functions primarily as:

• A regulator of maturation and trafficking of adenylyl cyclases (ACs) to primary cilia

• A repressor of the Hedgehog (Hh) signaling pathway via adenylyl cyclase targeting

• A potential player in neural tube development and patterning

Research has demonstrated that ANKMY2 is primarily a cytosolic protein that interacts with multiple adenylyl cyclases, determining their maturation and trafficking to primary cilia, which is crucial for proper cAMP signaling and morphogenetic patterning .

Which applications are ANKMY2 antibodies most commonly used for?

Based on the available data, ANKMY2 antibodies are primarily utilized in the following applications:

For Western blotting applications, ANKMY2 antibodies have been validated in multiple cell lines including HeLa cells, HepG2 cells, and mouse liver tissue, with an observed molecular weight of approximately 49 kDa . When performing IHC, ANKMY2 antibodies have been verified in human thyroid cancer and human gastric cancer samples .

What are the storage and handling recommendations for ANKMY2 antibodies?

For optimal performance and longevity of ANKMY2 antibodies, researchers should follow these standard protocols:

• Storage temperature: Store at -20°C (most manufacturers recommend avoiding freeze/thaw cycles)

• Storage buffer: Most ANKMY2 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Shipping conditions: Usually shipped with ice packs; upon receipt, store immediately at the recommended temperature

• Stability: Typically stable for 12 months when properly stored

• Aliquoting: Some manufacturers specifically recommend not to aliquot , while others suggest aliquoting to avoid repeated freeze-thaw cycles

CAUTION: Products containing sodium azide should be handled by trained staff only as it is a POISONOUS AND HAZARDOUS SUBSTANCE .

How should I design experiments to study ANKMY2's role in Hedgehog signaling?

When investigating ANKMY2's role in Hedgehog signaling, consider these validated experimental approaches:

Knockdown/Knockout Studies:

• RNA interference (RNAi) targeting ANKMY2 has been successfully used to demonstrate its function in regulating adenylyl cyclase localization and Hedgehog pathway activity

• For complete ablation of ANKMY2 function, genetic knockout models (as described in Somatilaka et al., 2020) can reveal dramatic phenotypes such as neural tube ventralization

Functional Analysis:

• Measure Hedgehog pathway activity using established readouts such as Gli1/Ptch1 expression via qRT-PCR following ANKMY2 manipulation

• Assess Gli3 processing (Gli3FL to Gli3R ratio) by Western blot to determine ANKMY2's impact on repressor formation

• Analyze ciliary localization of adenylyl cyclases using immunofluorescence microscopy with appropriate markers

Epistasis Analysis:

• Generate double knockouts (e.g., ANKMY2/Smoothened) to determine pathway hierarchy and dependency relationships

• Use pathway agonists (SAG, purmorphamine) or antagonists (cyclopamine, GANT61) to probe at which level ANKMY2 functions in the Hedgehog pathway

Critical controls should include rescue experiments with wild-type ANKMY2 and possibly domain mutants to determine which protein regions are essential for function .

What are the critical considerations when validating ANKMY2 antibody specificity?

Ensuring antibody specificity is crucial for reliable research outcomes. For ANKMY2 antibodies, consider implementing these validation strategies:

Genetic Approaches:

• Utilize ANKMY2 knockout or knockdown models as negative controls to confirm specificity

• Overexpression systems with tagged ANKMY2 can serve as positive controls

Multiple Antibody Validation:

• Compare results using antibodies from different vendors or those targeting different epitopes

• When possible, use antibodies raised against different species to confirm cross-reactivity claims

Application-Specific Controls:

• For Western blotting: Include molecular weight markers and confirm band size matches the predicted 49 kDa

• For IHC: Use appropriate tissue controls including known positive (e.g., thyroid cancer, gastric cancer) and negative tissues

• For all applications: Include secondary antibody-only controls to assess background

Orthogonal Method Confirmation:

• Verify protein expression with complementary techniques (e.g., mass spectrometry)

• Correlate protein detection with mRNA expression data

Several manufacturers provide validation data for their ANKMY2 antibodies in specific applications which can serve as reference points for expected results .

How can I optimize ANKMY2 antibody use for Western blotting in diverse sample types?

Optimizing Western blotting protocols for ANKMY2 detection requires consideration of several factors:

Sample Preparation:

• Cell lysates (HeLa, HepG2): Standard RIPA buffer with protease inhibitors works effectively

• Tissue samples (liver): More stringent extraction buffers may be necessary to fully solubilize membrane-associated ANKMY2

• Consider phosphatase inhibitors if studying potential post-translational modifications

Protocol Optimization:

• Loading: 20-40 μg total protein per lane is typically sufficient

• Separation: 10% SDS-PAGE gels provide optimal resolution for the 49 kDa ANKMY2 protein

• Transfer: Semi-dry or wet transfer at 100V for 60-90 minutes (use PVDF membrane for best results)

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature

• Primary antibody: Start with 1:500 dilution and titrate as needed

• Incubation: Overnight at 4°C generally yields the best signal-to-noise ratio

• Detection: HRP-conjugated secondary antibodies with ECL detection systems are suitable for most applications

Troubleshooting Common Issues:

• Multiple bands: May indicate splice variants, post-translational modifications, or non-specific binding

• Weak signal: Increase antibody concentration, extend incubation time, or use signal enhancement systems

• High background: Increase blocking stringency or washing steps

For quantitative analysis, ensure linear detection range by performing a dilution series of your sample and use appropriate housekeeping controls .

How can I investigate ANKMY2's interaction with adenylyl cyclases in ciliary trafficking?

ANKMY2's role in adenylyl cyclase (AC) trafficking to cilia requires specialized techniques:

Protein-Protein Interaction Analysis:

• Co-immunoprecipitation (co-IP): Successfully used to demonstrate ANKMY2 interaction with ADCY3, ADCY5, and ADCY6

• Tandem affinity purification followed by mass spectrometry (TAP-MS): Effectively identified Ankmy2 as an interaction partner of adenylyl cyclases

• Proximity labeling methods (BioID, APEX) can map the interaction landscape around ANKMY2 in relevant cellular compartments

Ciliary Localization Studies:

• Immunofluorescence microscopy with ciliary markers (acetylated tubulin, Arl13b) and adenylyl cyclase antibodies in wild-type vs. ANKMY2-depleted cells

• Live-cell imaging using fluorescently tagged ANKMY2 and adenylyl cyclases to track trafficking dynamics

• Super-resolution microscopy for detailed localization analysis within ciliary subcompartments

Functional Assays:

• FRET-based cAMP sensors to measure ciliary cAMP dynamics in response to ANKMY2 manipulation

• Ciliary length measurements as a phenotypic readout (ANKMY2 knockdown has been shown to reduce ciliary length by ~23%)

• Forskolin stimulation assays to assess cAMP accumulation in cilia with or without ANKMY2

Key experimental considerations include using adequate ciliary markers, ensuring proper cell cycle arrest to promote ciliogenesis, and implementing quantitative image analysis methods for reliable measurements .

What explains the seemingly contradictory findings regarding ANKMY2's role in Hedgehog signaling?

The literature reveals apparent contradictions regarding ANKMY2's role in Hedgehog signaling that require careful analysis:

Contradictory Findings:

• Somatilaka et al. (2020) demonstrated that ANKMY2 knockout causes increased Hedgehog pathway activity and neural tube ventralization, suggesting ANKMY2 is a pathway repressor

• Saita et al. (2014) reported that ANKMY2 positively regulates Hedgehog signaling in cultured cells

• Later studies showed that ANKMY2 knockdown reduces SHH-stimulated Gli1 induction in certain cell types, suggesting a positive regulatory role

Potential Explanations:

• Context-dependency: ANKMY2's function may differ between developmental contexts (embryonic neural tube) and adult tissues or cell culture models

• Cell-type specificity: Different cell types (NIH-3T3 vs. IMCD3) may have distinct requirements for ANKMY2 in Hedgehog signaling

• Temporal considerations: Acute (knockdown) versus chronic (knockout) loss of ANKMY2 may yield different phenotypes due to compensatory mechanisms

• Methodological differences: Varying assay sensitivities and experimental conditions can influence outcomes

Reconciliation Approach:
To address these contradictions, researchers should:

• Perform parallel experiments in multiple cell types using both knockdown and knockout approaches

• Assess both ligand-dependent and ligand-independent Hedgehog pathway activation

• Evaluate ANKMY2's role at different time points during development and in adult tissues

• Consider ANKMY2's interaction with other regulatory proteins (e.g., FKBP38)

The dual role of ANKMY2 suggests it may function in a context-specific manner, potentially switching between positive and negative regulation depending on cellular conditions .

How does ANKMY2 impact Gli protein processing and what techniques can best evaluate this mechanism?

ANKMY2 has been shown to significantly influence Gli protein processing, particularly affecting Gli repressor formation:

ANKMY2's Impact on Gli Processing:

• ANKMY2 knockout embryos exhibit reduced processing of Gli3 into Gli3R and Gli2 into Gli2R

• Gli3 full-length to Gli3R ratios are significantly increased in ANKMY2 knockout embryos

• Both Gli3 processing defects and increased Hedgehog signaling in ANKMY2 knockouts occur independently of Smoothened, suggesting direct regulation of Gli processing

Technical Approaches for Studying Gli Processing:

• Protein Analysis Techniques:

  • Western blotting with Gli2/Gli3-specific antibodies that detect both full-length and repressor forms

  • Co-immunoprecipitation to assess interactions between ANKMY2 and Gli proteins or processing machinery

  • Phosphorylation-specific antibodies to detect PKA-mediated phosphorylation of Gli proteins (a prerequisite for processing)

• Genetic and Cellular Approaches:

  • Epistasis experiments combining ANKMY2 manipulation with alterations in PKA, GSK3β, or CK1 activity

  • CRISPR-Cas9 editing of Gli phosphorylation sites to assess their requirement in ANKMY2-mediated processing

  • Subcellular fractionation to determine if ANKMY2 affects Gli protein localization

• Advanced Imaging Methods:

  • Live-cell imaging of fluorescently tagged Gli proteins to track processing dynamics

  • Proximity ligation assays to visualize interactions between ANKMY2 and Gli processing machinery in situ

When designing these experiments, researchers should consider that Gli3 processing is more robust and easily detected than Gli2 processing, making Gli3 often the preferred readout for initial studies .

What is the evidence for ANKMY2's involvement in pathological conditions?

Current research suggests ANKMY2 may be implicated in several pathological conditions:

Developmental Disorders:

• Complete loss of ANKMY2 causes embryonic lethality at mid-gestation with severe neural tube defects in mouse models

• The neural tube phenotype in ANKMY2 knockout mice resembles severe human neural tube defects, suggesting potential relevance to human congenital disorders

Cancer and Hematological Malignancies:

• Downregulation of ANKMY2, associated with frequent deletions of human chromosome 7p22.1, may play a role in the pathogenesis of natural killer (NK)-cell malignancies

• ANKMY2 is upregulated by enforced expression of Hox11, which functions to hinder hemopoiesis, diverts differentiation to an alternative fate and promotes pre-leukemic states

Hedgehog-Related Pathologies:

• Given ANKMY2's critical role in regulating Hedgehog signaling, it may have implications for Hedgehog-driven cancers (medulloblastoma, basal cell carcinoma) and developmental disorders

For clinical investigations, researchers should consider:

• Analyzing ANKMY2 expression in patient samples from relevant pathologies

• Correlating ANKMY2 levels with disease progression or treatment response

• Exploring genetic variations in ANKMY2 that might contribute to disease susceptibility

How can I design experiments to investigate ANKMY2's potential role in cancer?

To investigate ANKMY2's role in cancer, consider these methodological approaches:

Expression Analysis:

• Analyze ANKMY2 expression across cancer types using publicly available databases (TCGA, CCLE)

• Perform immunohistochemistry on cancer tissue microarrays using validated ANKMY2 antibodies (dilution 1:50-1:100)

• Conduct qRT-PCR and Western blot analysis on primary tumor samples compared to matched normal tissues

Functional Studies:

• In vitro approaches:

  • Modulate ANKMY2 levels in cancer cell lines through overexpression, knockdown, or CRISPR-Cas9 knockout

  • Assess effects on:

    • Cell proliferation, migration, and invasion assays

    • Hedgehog pathway activity using Gli1/Ptch1 expression as readouts

    • Cilia formation and function in ciliated cancer cells

  • Combine with Hedgehog pathway modulators to determine functional relationships

• In vivo approaches:

  • Generate xenograft models with ANKMY2-modulated cancer cells

  • Create conditional knockout mouse models targeting ANKMY2 in cancer-prone tissues

  • Evaluate tumor incidence, growth, and metastatic potential

Mechanistic Investigations:

• Explore ANKMY2's relationship with Hox11 in hematopoietic malignancies

• Investigate the effect of ANKMY2 on adenylyl cyclase localization and cAMP signaling in cancer cells

• Examine if ANKMY2 affects Gli protein processing and Hedgehog pathway output in cancer contexts

Key controls should include rescue experiments with wild-type ANKMY2 and correlation with established cancer drivers in the experimental model systems.

What approaches can be used to study ANKMY2's role in normal and pathological development?

ANKMY2's critical role in development, particularly in neural tube formation, can be studied using various approaches:

Developmental Model Systems:

• Mouse embryos: The established ANKMY2 knockout mouse model shows complete neural tube ventralization and embryonic lethality

• Zebrafish: Targeting the zebrafish ortholog (ankmy2a) provides a complementary vertebrate model with advantages for real-time imaging

• Ex vivo organ cultures: Neural tube explants can be manipulated to study ANKMY2 function in a more controlled environment

Temporal and Spatial Expression Analysis:

• In situ hybridization to map ANKMY2 expression patterns throughout development

• Immunohistochemistry with anti-ANKMY2 antibodies (1:50-1:100 dilution) to localize protein expression

• Single-cell RNA sequencing to identify cell populations with enriched ANKMY2 expression

Functional Developmental Studies:

• Conditional knockout approaches:

  • Tissue-specific Cre lines (e.g., Sox2-Cre for epiblast deletion)

  • Tamoxifen-inducible CreERT2 systems for temporal control

  • CRISPR-Cas9 tissue-specific editing using appropriate promoters

• Rescue experiments:

  • Wild-type ANKMY2 expression in knockout backgrounds

  • Domain-specific mutants to identify critical functional regions

  • Cross-species rescue to assess functional conservation

• Pathway analysis:

  • Assess neural progenitor markers (FoxA2, Nkx2.2, Olig2, Nkx6.1, Pax6) to evaluate dorsal-ventral patterning

  • Combine with Hedgehog pathway modulation (Smoothened agonists/antagonists)

  • Evaluate Gli repressor formation in developing tissues

For pathological developmental contexts, researchers should consider examining ANKMY2 expression and genetic variations in human developmental disorders, particularly neural tube defects .