atoh8 Antibody

What is ATOH8 and why is it important in research?

ATOH8 (also known as MATH6, ATH6, or HATH6) is a bHLH transcription factor that exhibits 43-57% identity in the bHLH domain with other mammalian atonal paralogs including NeuroD and Neurogenin factors . It is indispensable for early embryonic development and participates in tissue-specific differentiation processes.

Research indicates ATOH8 is critically involved in:

• Myogenesis and skeletal muscle maintenance

• Osteoclastogenesis regulation through BMP signaling

• Hypoxic response attenuation via HIF-2α regulation

• Tumor suppression in lung cancer models

• Immune regulation in hepatitis B virus infection

• Intestinal microfold cell differentiation

What detection methods are compatible with commercial ATOH8 antibodies?

Based on validated research applications, ATOH8 antibodies can be applied to multiple detection methodologies:

For optimal results, antigen retrieval with citrate buffer (pH 6.0, 121°C for 5 min) is recommended for IHC applications .

What molecular weight should be detected for ATOH8 in Western blots?

ATOH8 has a calculated molecular weight of approximately 35 kDa . When performing Western blot analysis, researchers should expect to observe a band at this molecular weight. Differences in observed molecular weight may occur due to:

• Post-translational modifications

• Protein degradation during sample preparation

• Splice variants

• Non-specific binding

Validation with positive and negative controls is essential for confirming antibody specificity.

How should researchers validate ATOH8 antibody specificity?

Thorough validation is critical for obtaining reliable results with ATOH8 antibodies:

• Genetic controls: Compare tissues/cells from wild-type vs. Atoh8-knockout models. Peyer's patches from intestinal-specific Atoh8 knockout mice (Vil1-cre;Atoh8-floxed) provide excellent negative controls .

• Overexpression systems: Use ATOH8-Flag overexpressing cell lines (e.g., C2C12-OE) to confirm antibody specificity .

• Immunohistochemistry validation: Confirm tissue-specific expression patterns. For example, ATOH8 should be detectable in:

  • Osteoblasts lining trabecular bone surfaces (but not in osteocytes)

  • Skeletal muscles

  • Prehypertrophic chondrocytes

• Antibody selection: Choose antibodies targeting conserved epitopes when working with multiple species. The immunogen sequence "MKHIPVLEDGPWKTVCVKELNGLKKLKRKGKEPARRANGYKTFRLDLEAPEPRAVATNGLRDRTHRLQPVPVPVPVPVP" shows high conservation (73% mouse, 72% rat identity).

What methodological challenges exist in detecting ATOH8 in different tissue contexts?

Several tissue-specific challenges require optimization:

• Low endogenous expression: In some cell types like HepG2.2.15, endogenous ATOH8 expression is extremely low , necessitating sensitive detection methods.

• Tissue-specific expression patterns: In adult mice, ATOH8 is detectable in osteoblasts but not in osteocytes . During development (E17.5), it's expressed in prehypertrophic chondrocytes and skeletal muscles adjacent to bone, but not in embryonic osteoblasts .

• Technical limitations: Some commercial ATOH8 antibodies are not suitable for co-immunoprecipitation experiments , limiting protein interaction studies.

• Temporal expression variances: ATOH8 expression changes during differentiation. For example, during myoblast differentiation, expression levels vary significantly between proliferative phase (Day 0) and differentiation days 1-3 .

How can ATOH8 expression be effectively manipulated in experimental models?

Several validated approaches for modulating ATOH8 expression:

• Genetic knockout models:

  • Global Atoh8-knockout mice show mild phenotypes in neonate skeletons but significant bone volume decrease and osteoclast increases in adulthood .

  • Tissue-specific conditional knockout using Cre-lox system (e.g., Vil1-cre;Atoh8-floxed for intestinal studies) .

• Viral vector overexpression:

  • Retrovirus carrying Atoh8-Flag for stable overexpression in cell lines (e.g., C2C12 myoblasts) .

  • Recombinant lentiviral vectors (e.g., pHBLV-CMVIE-ZsGreen-Puro) for ATOH8 overexpression in hepatoma cell lines .

• siRNA knockdown:

  • Validated siRNA approaches show enhanced expression of HIF-2α target genes (DLL4, ANGPT2) in human pulmonary artery endothelial cells .

• BMP pathway modulation:

  • BMP-6 (100 ng·mL−1) significantly increases Atoh8 expression in ST-2 stromal cells and MC3T3-E1 osteoblasts at 48h .

  • LDN193189 (BMP type I receptor inhibitor) blocks BMP-6-induced Atoh8 expression .

How is ATOH8 expression regulated at the genomic level?

ATOH8 expression is regulated through several mechanisms:

• BMP-Smad1 signaling axis:

  • ChIP-seq and ChIP-qPCR analyses have identified Smad1/5-binding regions (SBRs) in the Atoh8 gene locus .

  • BMP-6 (100 ng/mL) significantly increases Atoh8 expression in osteoblasts, with effects blocked by BMP type I receptor inhibitor LDN193189 .

• DNA methylation:

  • Hypermethylation of the ATOH8 gene occurs in approximately 12% of lung adenocarcinoma (LUAD) cases .

  • Methylation silencing is comparable to well-established tumor suppressor genes CDKN2A and RASSF1 .

  • Methylation status correlates with extremely low endogenous ATOH8 levels in certain cancer cell lines (A549, PC9) .

• Tissue-specific transcriptional regulators:

  • ATOH8 is part of the pancreatic transcriptional network during embryonic development .

  • It may function as a modulator of differentiation programs initiated by the pro-endocrine factor Neurog3 .

What protein interactions mediate ATOH8's biological functions?

ATOH8 interacts with multiple proteins to exert its biological effects:

• Transcription factor interactions:

  • ATOH8 forms protein complexes with RUNX2 to inhibit RUNX2 transcriptional activity and decrease the RANKL/OPG expression ratio in osteoblasts .

• HIF pathway regulation:

  • The bHLH domain of ATOH8 (specifically amino acids 241-289) is necessary and sufficient for interaction with and degradation of HIF-1α and HIF-2α .

  • FLAG-ATOH8 co-precipitates with endogenous HIF-2α in stably-expressing HPAECs .

  • ATOH8 attenuates hypoxia-induced HIF-2α activation and selectively suppresses HIF-2α target genes (DLL4, ANGPT2) without affecting HIF-1α-selective targets (VEGFA, PGK1) .

• SMAD3 binding:

  • ATOH8 binds SMAD3 to induce cellular senescence and consequent tumor suppression in lung cancer models .

  • SMAD3 inhibitors can delay spontaneous lung tumor formation in mouse models driven by mutant Ras and Atoh8 depletion .

What is known about ATOH8's role in pulmonary diseases and hypoxic responses?

ATOH8 plays critical roles in pulmonary pathophysiology:

• Pulmonary arterial hypertension (PAH):

  • Atoh8-deficient mice exhibit PAH-like pathological changes including increased Fulton index and coverage of peripheral small arteries by SMA-positive cells .

  • These phenotypes resemble mice with dysregulated BMPRII/ALK-1/SMAD pathway .

  • The ALK-1/SMAD/ATOH8 axis attenuates hypoxic responses in the lung vasculature.

• Lung cancer suppression:

  • ATOH8 functions as a potential noncanonical tumor suppressor without significant genetic mutations .

  • ATOH8 gene hypermethylation occurs in 11.8% of lung adenocarcinoma cases .

  • ATOH8-mediated tumor suppression involves SMAD3 binding and cellular senescence induction .

How does ATOH8 influence inflammatory and immune responses?

ATOH8 modulates immune functions in several contexts:

• HBV immune tolerance:

  • ATOH8 overexpression in HepG2.2.15 cells increases HBV DNA levels and HBsAg expression .

  • ATOH8 inhibits pyroptosis in hepatocytes, potentially assisting HBV in immune escape by inhibiting inflammatory pathway activity .

  • ATOH8 overexpression increases secretion of INF-α and TNF-α by HepG2.2.15 cells but shows complex effects on pyroptosis-related cytokines IL-18 and IL-1β .

• Intestinal immunity:

  • ATOH8 regulates intestinal microfold cell (M cell) differentiation .

  • Atoh8 deficiency leads to increased Gp2 expression in intestinal epithelia through epithelium-intrinsic mechanisms demonstrated in RankL-treated organoids .

What methodological approaches are best for studying ATOH8 in muscle development and maintenance?

Specialized techniques for investigating ATOH8 in muscle contexts:

• Proliferation assessment:

  • BrdU incorporation assay (10 μM for 3-10 hours) followed by anti-BrdU immunostaining provides quantifiable proliferation data in both primary myoblasts and cell lines .

  • C2C12-OE cells (stably overexpressing Atoh8-Flag) show significantly higher proliferation rates compared to control C2C12 cells .

• Differentiation analysis:

  • Immunostaining for differentiation markers:

    • Desmin for myoblast identity verification

    • Myosin heavy chain 2 (MYH2) for myotube formation assessment

  • Myogenic fusion index calculation to quantify differentiation potential .

  • qPCR analysis of myogenic regulatory factors (MRFs) to track differentiation progression .

• In vivo phenotyping:

  • Body weight comparisons between wild-type and Atoh8 knockout mice at 3 months .

  • Histological analysis of muscle tissue to assess fiber type composition and organization.

What are the optimal parameters for Western blot detection of ATOH8?

When performing Western blot analysis for ATOH8:

• Sample preparation:

  • Standard lysis buffers containing protease inhibitors are suitable.

  • For comparing ATOH8 protein levels in different experimental conditions, ensure equal loading by total protein normalization or housekeeping protein controls.

• Expected molecular weight:

  • ATOH8 has a calculated molecular weight of 35 kDa .

  • Verify antibody specificity with appropriate positive and negative controls.

• Protein degradation considerations:

  • The proteasome inhibitor MG132 can rescue ATOH8-induced degradation of target proteins like HIF-2α , suggesting ATOH8 itself may be subject to proteasomal degradation.

How can researchers quantify changes in ATOH8 expression in response to experimental treatments?

Several validated approaches for quantifying ATOH8 expression changes:

• qPCR for transcript analysis:

  • Used successfully to measure Atoh8 mRNA expression during myoblast differentiation stages .

  • Effective for quantifying BMP-induced Atoh8 expression in osteoblasts .

• Western blot for protein quantification:

  • Densitometric analysis of 35 kDa ATOH8 band normalized to loading controls.

  • Used to confirm ATOH8 overexpression in engineered cell lines .

• Immunohistochemistry for tissue expression:

  • RNAscope in situ hybridization effectively detects Atoh8 expression in muscle and bone tissues .

  • Counterstaining with DAPI helps calculate percentage of ATOH8-positive cells.

• Reporter systems:

  • Fluorescent markers like ZsGreen in pHBLV-CMVIE-ZsGreen-Puro vector system allow visualization of successful ATOH8 expression .

  • Puromycin selection ensures population of ATOH8-expressing cells for analysis .

What are best practices for immunohistochemical and immunofluorescent detection of ATOH8?

For optimal immunostaining results:

• Antigen retrieval:

  • Citrate buffer, pH 6.0 (121°C for 5 min) is effective for ATOH8 detection in tissue sections .

• Blocking conditions:

  • 1% PBS/BSA supplemented with 5% normal donkey serum reduces background .

• Antibody incubation:

  • Overnight incubation at 4°C with primary ATOH8 antibody yields optimal results .

• Control tissues:

  • Positive controls: Adult mouse trabecular bone (osteoblasts), skeletal muscle .

  • Negative controls: Osteocytes embedded in bone matrix .

• Detection systems:

  • Standard secondary antibody systems work well for both chromogenic and fluorescent detection.

  • Include appropriate negative controls (isotype control or secondary antibody only).