ANKRD26 Antibody

What is ANKRD26 and why is it important in research?

ANKRD26 is a 192-196 kDa protein encoded by the ANKRD26 gene located on chromosome 10p12.1. It contains spectrin helices and ankyrin repeats, protein domains known to interact with cytoskeletal and signaling proteins .

ANKRD26 is critically important in research for several reasons:

• It serves as the ancestral gene for the POTE (Prostate-, Ovary, Testis-, and placenta-Expressed) family of primate-specific genes

• Germline mutations in the 5' UTR regulatory region are associated with thrombocytopenia 2 (THC2), an inherited platelet disorder with predisposition to hematological malignancies

• It plays crucial roles in modulating cytokine-dependent signaling pathways, particularly in hematopoiesis

• It forms organizational nanoclusters at the plasma membrane that are important for cellular differentiation and signaling

ANKRD26 expression is tightly regulated during hematopoiesis, with high expression in hematopoietic stem cells and downregulation during megakaryopoiesis, becoming almost undetectable in mature megakaryocytes and platelets .

What are the key structural and functional domains of ANKRD26 relevant for antibody selection?

ANKRD26 contains several key structural domains that are important considerations when selecting antibodies:

The antibody selection should be guided by the specific protein region you wish to target. For instance:

• C-terminal antibodies (e.g., 20035-1-AP) target the region beyond amino acid 1650

• N-terminal antibodies (e.g., ab47984) target within the first 50 amino acids

When studying membrane localization or AML-associated mutations, antibodies targeting the N-terminal region would be most informative, while antibodies targeting the C-terminal region may be better for studying self-association properties .

What are the recommended applications for ANKRD26 antibodies in research?

Based on validated research applications, ANKRD26 antibodies can be reliably used in the following applications:

For studying ANKRD26 nanoclusters at the plasma membrane, freeze-fracture immunogold electron microscopy has proven particularly effective, allowing visualization of self-associated ANKRD26 complexes spanning 5-40 nm with clusters containing up to 17 molecules .

How can I optimize Western blot protocols for ANKRD26 detection?

Optimizing Western blot detection of ANKRD26 requires careful consideration due to its high molecular weight (~196 kDa):

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors

  • Include phosphatase inhibitors when studying phosphorylation-dependent signaling

  • Heat samples at 70°C for 10 minutes rather than 95°C to prevent aggregation of large proteins

• Gel electrophoresis:

  • Use low percentage (6-8%) SDS-PAGE gels or gradient gels (4-15%)

  • Extend running time to ensure adequate separation of high molecular weight proteins

  • Include ladder markers that extend beyond 200 kDa

• Transfer conditions:

  • Implement wet transfer at 30V overnight at 4°C for large proteins

  • Use 0.45 μm PVDF membrane rather than 0.2 μm for better retention of large proteins

• Antibody selection and dilution:

  • For human samples, validated antibodies include 20035-1-AP and ab86780

  • Begin with manufacturer's recommended dilution and optimize as needed

  • Extended primary antibody incubation (overnight at 4°C) improves detection

• Controls:

  • Include positive controls such as cells with known ANKRD26 expression

  • RNAi knockdown samples serve as negative controls

  • Consider using recombinant ANKRD26 fragments as additional controls

When investigating ANKRD26's role in signaling pathways, examining its effects on downstream targets like ERK1/2, STAT3, and AKT phosphorylation provides valuable insights into its functional significance .

What methodologies are effective for studying ANKRD26 interactions with cytokine receptors?

ANKRD26 has been shown to interact with and modulate the activity of homodimeric type I cytokine receptors including MPL, EPOR, and G-CSFR . The following methodologies have proven effective for studying these interactions:

• Co-immunoprecipitation (Co-IP):

  • Transfect cells with tagged versions of ANKRD26 and receptor of interest

  • Use reciprocal pull-downs (e.g., pull down ANKRD26 and blot for receptor, and vice versa)

  • Example: HEK293T cells transfected with myc-ETV6 and FLAG-ANKRD26 showed clear interaction

• Proximity Ligation Assay (PLA):

  • Allows detection of protein interactions in situ with high sensitivity

  • Use antibodies against ANKRD26 and the receptor of interest

  • Signals appear as fluorescent dots where proteins are in close proximity (<40 nm)

  • Successfully used to detect ANKRD26-ETV6 interactions

• Receptor internalization assays:

  • Surface biotinylation followed by internalization at various timepoints

  • Flow cytometry with receptor-specific antibodies

  • Studies have shown ANKRD26 prevents receptor internalization, leading to increased signaling

• Functional signaling assays:

  • Luciferase reporter assays (e.g., MMP3 promoter activity for ETV6 function)

  • Phospho-specific Western blots targeting downstream pathways (ERK1/2, STAT3, AKT)

  • Ba/F3 cell lines expressing specific receptors with/without ANKRD26 overexpression

• Cytokine sensitivity assays:

  • Dose-response curves with increasing concentrations of ligands (TPO, EPO, G-CSF)

  • Measurement of proliferation, differentiation, or signaling activation

  • ANKRD26 overexpression increases cytokine sensitivity across multiple receptor systems

These approaches have demonstrated that higher than normal levels of ANKRD26 prevent receptor internalization, which leads to increased signaling and cytokine hypersensitivity .

How can I effectively analyze ANKRD26 expression during hematopoietic differentiation?

Analyzing ANKRD26 expression during hematopoietic differentiation requires careful consideration of its dynamic regulation. The following methodological approaches have proven effective:

• Cell model selection:

  • Primary CD34+ progenitor cells from cord blood or bone marrow

  • Patient-derived induced pluripotent stem cells (iPSCs)

  • Cell lines modeling specific lineages (e.g., DAMI for megakaryocytes, UT7 for erythroid)

• Differentiation protocols:

  • For megakaryocytic differentiation: TPO (10-100 ng/ml)

  • For erythroid differentiation: EPO and SCF

  • For granulocytic differentiation: G-CSF, SCF, and IL-3

• Expression analysis techniques:

  • qRT-PCR for mRNA expression levels normalized to appropriate housekeeping genes

  • Western blot for protein expression at different differentiation stages

  • Immunofluorescence to visualize subcellular localization changes

• Genetic manipulation approaches:

  • Lentiviral overexpression of ANKRD26 to study gain-of-function effects

  • shRNA or siRNA for knockdown studies (validated sequences available in literature)

  • CRISPR/Cas9 for generating knockout models

• Functional readouts:

  • Colony-forming assays in methylcellulose (CFU-G, CFU-E, CFU-Meg)

  • Proplatelet formation for megakaryocytes

  • Cell proliferation and differentiation marker expression

Research has shown that ANKRD26 expression is high in early progenitors and progressively silenced during differentiation in all three myeloid lineages (erythroid, megakaryocytic, and granulocytic). Failure to downregulate ANKRD26 in THC2 patients impairs differentiation, particularly affecting proplatelet formation in megakaryocytes .

How do mutations in the ANKRD26 gene affect antibody-based detection methods?

Mutations in ANKRD26, particularly those associated with THC2 and hematological malignancies, can impact antibody-based detection in several important ways:

• 5′ UTR mutations (c.-116 to c.-134):

  • Most common in THC2 patients

  • Don't affect protein coding sequence, so antibody epitopes remain intact

  • Result in sustained expression of ANKRD26 during megakaryopoiesis

  • Detection requires antibodies that can distinguish expression patterns rather than protein structure

  • May require custom panel designs that include the 5′ UTR as a known variant hotspot

• AML-associated N-terminal mutations:

  • Affect the membrane-binding N-terminal amphipathic structure

  • May alter subcellular localization from membrane to cytosol

  • Antibodies targeting N-terminal epitopes may show reduced binding

  • Immunofluorescence will show altered localization patterns

• WAC-ANKRD26 fusion transcripts:

  • Rare paired-duplication inversion resulting in fusion protein

  • May require specialized detection methods beyond conventional antibodies

  • Western blots would show band at unexpected molecular weight

  • May require combined RNA-seq and long-read genome sequencing for identification

• Copy number variations:

  • Approximately 30% of RUNX1 cases harbor deletions

  • May not be detectable with standard antibody techniques

  • Require genome array technology or specialized bioinformatic pipelines

  • May present as reduced signal intensity in quantitative immunoassays

For research involving THC2 patients, it's essential to use antibodies that can detect the wild-type ANKRD26 protein while being aware that the pathology stems from dysregulated expression rather than structural protein changes. When studying hematological malignancies, combining antibody-based detection with genetic sequencing provides the most comprehensive analysis .

What functional assays can determine the pathogenicity of ANKRD26 variants?

Determining the pathogenicity of ANKRD26 variants requires functional assays that assess their impact on key cellular processes. The following approaches have proven valuable:

• Luciferase-based transcriptional assays:

  • Successfully established for ANKRD26, similar to validated assays for RUNX1 and ETV6

  • Measures impact of 5′ UTR mutations on gene expression regulation

  • Can help resolve variants of uncertain significance (VUSs)

  • Currently being assessed for clinical use in diagnosing THC2

• Megakaryocyte differentiation and proplatelet formation:

  • CD34+ cells transduced with wild-type or mutant ANKRD26

  • Assessment of megakaryocyte proliferation and maturation

  • Quantification of proplatelet-forming megakaryocytes

  • THC2-associated variants completely prevent proplatelet formation

• MAPK/ERK1/2 signaling assays:

  • Measurement of TPO-dependent ERK1/2 phosphorylation

  • THC2 mutations lead to increased ERK phosphorylation and elevated MAPK signaling

  • Sustained activation prevents platelet formation

• Receptor internalization and trafficking:

  • Surface biotinylation and internalization kinetics

  • Flow cytometry measuring receptor surface expression

  • Wild-type ANKRD26 should be downregulated during differentiation, whereas pathogenic variants lead to sustained expression and receptor hypersensitivity

• Colony-forming unit (CFU) assays:

  • Semi-solid methylcellulose culture systems

  • Assessment of lineage-specific colony formation (CFU-G, CFU-Meg)

  • THC2 patients show increased granulocyte colony numbers (>2-fold) and altered differentiation patterns

For clinical variant classification, high-throughput screening technologies like multiplexed assays of variant effect could potentially generate functional data for all possible missense variants, similar to approaches used for other cancer predisposition genes like TP53 and BRCA1 .

How does ANKRD26 contribute to hematological malignancy development?

ANKRD26 mutations contribute to hematological malignancy development through several mechanisms:

• Dysregulated signaling pathways:

  • Sustained ANKRD26 expression prevents normal downregulation during differentiation

  • Leads to hyperactivation of MAPK/ERK1/2 signaling

  • Results in cytokine hypersensitivity and altered proliferation/differentiation balance

  • Similar mechanisms seen with other leukemia predisposition genes (RUNX1, ETV6)

• Impaired cellular differentiation:

  • Prevents completion of terminal differentiation in myeloid lineages

  • Creates expanded pool of progenitor cells susceptible to additional mutations

  • ANKRD26 knockdown studies show it's critical for cellular differentiation processes

• Abnormal receptor trafficking and signaling:

  • Prevents internalization of cytokine receptors (MPL, EPOR, G-CSFR)

  • Leads to prolonged signaling and cytokine hypersensitivity

  • Creates persistent proliferative signals

• Disruption of centriole copy number regulation:

  • ANKRD26 is involved in maintaining centriole copy number

  • Dysregulation affects megakaryocyte polyploidization

  • May impact chromosomal stability over time

• Interaction with other leukemia-associated transcription factors:

  • Forms complexes with ETV6 (another leukemia predisposition gene)

  • ANKRD26 can retain ETV6 in the cytoplasm, preventing its nuclear function

  • Interacts with GPS2, affecting the interplay between ANKRD26 and ETV6

Clinical evidence shows THC2 patients have an increased risk of developing hematological malignancies, particularly acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The incidence of leukemia development in THC2 families is notably higher than in the general population .

How can ANKRD26 antibodies be employed in studying membrane nanocluster formation?

ANKRD26 forms dynamic nanoclusters at the plasma membrane that are critical for its function. Studying these structures requires specialized techniques:

• Freeze-fracture immunogold electron microscopy:

  • Gold standard for detecting endogenous ANKRD26 nanoclusters

  • Allows visualization of clusters ranging from 5-40 nm

  • Can detect superclusters containing up to 17 ANKRD26 molecules

  • Enables quantitative analysis of cluster distribution and density

  • Provides evidence of ANKRD26 insertion into the cytosolic leaflet of plasma membranes

• Super-resolution microscopy approaches:

  • STORM (Stochastic Optical Reconstruction Microscopy)

  • PALM (Photoactivated Localization Microscopy)

  • SIM (Structured Illumination Microscopy)

  • Requires careful antibody selection and validation for these techniques

• FRET-based approaches:

  • Fluorescence Resonance Energy Transfer to detect protein proximity

  • Requires dual labeling with compatible fluorophore pairs

  • Can be combined with FLIM (Fluorescence Lifetime Imaging Microscopy)

  • Useful for studying dynamic associations in living cells

• Biochemical membrane fractionation:

  • Isolation of detergent-resistant membrane fractions

  • Differential centrifugation to separate membrane compartments

  • Western blot analysis of ANKRD26 distribution in different fractions

  • Successfully demonstrated ANKRD26 enrichment in membrane fractions

• Liposome binding assays:

  • In vitro reconstitution using purified recombinant ANKRD26

  • Assessment of binding to artificial membrane systems

  • Allows testing of specific lipid compositions and membrane curvatures

  • Demonstrated that the N-Ank module is critical for membrane association

When designing experiments to study ANKRD26 nanoclusters, it's important to note that about one-third of ANKRD26 molecules exist as single entities, another third as doublets, and the final third in clusters of three or more, with some superclusters extending up to ~200 nm and containing up to 17 molecules .

What approaches can resolve contradictory data regarding ANKRD26 localization and function?

The literature contains some apparent contradictions regarding ANKRD26 localization and function, particularly between its reported roles at the plasma membrane versus the centrosome. The following approaches can help resolve these discrepancies:

• Multi-modal localization studies:

  • Combine immunofluorescence with biochemical fractionation

  • Use multiple validated antibodies targeting different epitopes

  • Employ super-resolution microscopy to distinguish closely positioned structures

  • Perform co-localization with both plasma membrane and centrosomal markers simultaneously

• Context-dependent analysis:

  • Examine localization across different cell types relevant to disease (megakaryocytes, myeloid progenitors)

  • Track localization through differentiation stages

  • Assess localization under different stimulation conditions (resting vs. cytokine-activated)

• Dynamic protein tracking:

  • Live-cell imaging with fluorescently tagged ANKRD26

  • FRAP (Fluorescence Recovery After Photobleaching) to assess mobility

  • Photoactivatable constructs to track protein movement between compartments

• Function-specific assays:

  • Perform domain-specific mutational analysis

  • Create chimeric proteins with domain swaps

  • Design rescue experiments with targeted localization signals

• Integrated multi-omics approaches:

  • Combine proteomics of different cellular compartments

  • Correlate with transcriptomics data from differentiation models

  • Integrate with interaction network mapping

The literature indicates that ANKRD26 is not exclusively localized to one compartment. While some studies emphasize its centrosomal role in centriole copy number regulation and PIDDosome activation , others demonstrate its presence throughout wide areas of the plasma membrane where it regulates receptor trafficking and signaling . Both locations may be physiologically relevant, with ANKRD26 potentially shuttling between compartments or performing distinct functions at each location depending on cellular context .

How can ANKRD26 antibodies contribute to developing therapeutic strategies for associated disorders?

ANKRD26 antibodies can play crucial roles in developing therapeutic strategies for THC2 and associated hematological malignancies:

• Target identification and validation:

  • Use antibodies to map critical functional domains

  • Identify protein-protein interaction surfaces amenable to therapeutic targeting

  • Validate the role of ANKRD26 in patient-derived samples

  • Research has identified the 5′ UTR, N-terminal membrane-binding domain, and receptor interaction domains as potential targets

• Screening assay development:

  • Create high-throughput screening platforms using ANKRD26 antibodies

  • Develop ELISA or AlphaScreen assays to measure protein-protein interactions

  • Design cell-based reporter systems to monitor ANKRD26-dependent signaling

  • Screen for compounds that restore normal ANKRD26 downregulation during differentiation

• Mechanism-based therapeutic approaches:

  • Target the MAPK pathway, which is hyperactivated in THC2

  • Develop strategies to restore receptor internalization

  • Design therapies that compensate for dysregulated megakaryopoiesis

  • Research suggests inhibition of the MAPK pathway could potentially rescue thrombocytopenia

• Biomarker development:

  • Use antibodies to monitor ANKRD26 expression as a predictive biomarker for leukemia development in THC2 patients

  • Develop immunoassays for early detection of abnormal myelopoiesis

  • Create tools for treatment response monitoring

• Personalized medicine approaches:

  • Characterize variant-specific effects using antibody-based functional assays

  • Develop variant-specific therapeutic strategies

  • Create patient stratification methods based on ANKRD26 expression patterns

  • International initiatives on rare diseases are working to develop appropriate models and therapies for preclinical testing and clinical trials

For leukemia prevention, the integration of genetic testing with functional assessments using ANKRD26 antibodies could help identify high-risk individuals and guide preemptive therapeutic interventions before malignant transformation occurs .