AMOTL1 Antibody

What is AMOTL1 and why is it significant for research?

AMOTL1 (angiomotin-like protein 1) is a peripheral membrane protein that functions as a component of tight junctions. It plays crucial roles in regulating cell polarity, paracellular permeability, and cell migration . AMOTL1 is particularly significant for research because:

• It inhibits the Wnt/beta-catenin signaling pathway by recruiting CTNNB1 to recycling endosomes, preventing its nuclear translocation

• It functions in the Hippo signaling pathway through interaction with YAP1, influencing cell proliferation and tissue growth

• Mutations in AMOTL1, particularly affecting amino acids 157-161, are associated with a newly defined orofacial clefting syndrome with congenital heart disease and other manifestations

• It has been implicated in various cancers, including gastric and cervical cancer, making it a potential therapeutic target

The protein has a calculated and observed molecular weight of 107 kDa, with the gene located at chromosome 11q21 .

What are the optimal storage and handling conditions for AMOTL1 antibodies?

For maintaining optimal antibody performance, follow these research-validated protocols:

Small volume options (20μl) often contain 0.1% BSA as a stabilizer . For experimental reproducibility, document the specific lot number and storage duration in your lab notebook.

What dilutions and applications are recommended for AMOTL1 antibodies?

AMOTL1 antibodies have been validated for multiple applications with specific optimal dilutions:

Remember that optimal dilutions may be sample-dependent. It is always recommended to titrate the antibody in your specific experimental system to obtain optimal results . Use positive controls such as HeLa cells, which are known to express AMOTL1, to validate your protocols .

How can I detect AMOTL1 and YAP1 interactions in cellular contexts?

To investigate the critical AMOTL1-YAP1 interaction that has been implicated in cancer progression, employ these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use anti-AMOTL1 antibody for immunoprecipitation followed by western blotting with anti-YAP1 antibody (or vice versa)

  • Include appropriate controls: IgG control, input control, and lysates from cells where either protein is knocked down

• Cyclohexamide (CHX) chase assay for protein stability studies:

  • Treat cells with CHX to inhibit protein synthesis

  • Collect cell lysates at different time points (0, 2, 4, 6, 8 hours)

  • Perform western blot analysis to detect both AMOTL1 and YAP1 levels

  • This approach can demonstrate how AMOTL1 extends YAP1's half-life by preventing its degradation

• Ubiquitination assays:

  • Transfect cells with HA-tagged ubiquitin (HA-Ub)

  • Treat with proteasome inhibitor MG132

  • Perform immunoprecipitation with anti-YAP1 antibody

  • Detect ubiquitination levels by western blot using anti-HA antibody

  • Compare conditions with varying AMOTL1 expression levels to assess how AMOTL1 influences YAP1 ubiquitination

• Subcellular fractionation:

  • Separate nuclear and cytoplasmic fractions

  • Perform western blot analysis to detect YAP1 localization under conditions of AMOTL1 overexpression or knockdown

  • This approach can demonstrate AMOTL1's role in promoting YAP1 nuclear translocation

Research has shown that AMOTL1 and YAP1 protect each other from ubiquitin-mediated degradation, and AMOTL1 promotes YAP1 translocation into the nucleus to activate downstream expression of genes like CTGF .

What experimental designs are most effective for studying AMOTL1's role in cancer models?

Based on published research methodologies, effective experimental approaches include:

• In vitro functional assays following AMOTL1 knockdown/overexpression:

  • Cell proliferation: MTT assay or EdU incorporation assay (analyze at 0, 24, 48, 72, and 96 hours)

  • Colony formation: Monolayer colony formation assay to assess clonogenic potential

  • Cell invasion: Transwell invasion assays with Matrigel coating

  • Cell cycle analysis: Flow cytometry with PI staining; confirm with western blot for cell cycle markers (p21/p27, pRb)

  • Apoptosis assessment: 7AAD and Annexin V double staining to distinguish early and late apoptosis

• Signaling pathway analysis:

  • MAPK pathway: Assess pERK1/2 levels after serum stimulation in cells with AMOTL1 knockdown compared to controls

  • Hippo pathway: Examine nuclear YAP1 localization and CTGF expression

  • Wnt/β-catenin pathway: Monitor β-catenin nuclear translocation

• In vivo tumor models:

  • Generate CRISPR/Cas9-based stable cell lines with AMOTL1 knockout

  • Perform xenograft experiments in immunodeficient mice

  • Monitor tumor growth, weight, and analyze tumor tissues by immunohistochemistry for AMOTL1, YAP1, CTGF, Ki67 (proliferation marker), and cleaved-Caspase 3 (apoptosis marker)

• Drug sensitivity testing:

  • Evaluate how AMOTL1 expression affects response to therapeutic agents

  • Consider verteporfin (VP), which targets YAP-TEAD interactions, and observe effects on AMOTL1 expression

Research has demonstrated that AMOTL1 knockdown significantly inhibits cancer cell proliferation, colony formation, and invasion capabilities while inducing cell cycle arrest and apoptosis .

How can I validate and optimize AMOTL1 antibody specificity for my specific research model?

To ensure high specificity and reliable results, implement these validation strategies:

• Multiple antibody approach:

  • Use antibodies targeting different epitopes of AMOTL1 (N-terminal, C-terminal, internal regions)

  • Compare results to confirm consistent detection patterns

• Genetic validation:

  • Employ AMOTL1 knockdown (siRNA) or knockout (CRISPR/Cas9) controls

  • Confirm decreased signal in western blot, IHC, or IF applications

  • Include rescue experiments by re-expressing AMOTL1 to restore signal

• Species cross-reactivity assessment:

  • For studies using non-human models, conduct sequence alignment analysis between human AMOTL1 and your species of interest

  • Predicted reactivity based on sequence homology: Cow (93%), Dog (79%), Guinea Pig (100%), Mouse (100%), Rabbit (100%)

  • Perform pilot tests in your specific model organism if the antibody hasn't been validated for that species

• Positive control selection:

  • Use cell lines known to express AMOTL1 (HeLa, SH-SY5Y, AGS, MKN28, BGC-823)

  • Include tissue samples with documented AMOTL1 expression (e.g., human prostate carcinoma tissue)

• Antibody titration:

  • Test a range of dilutions around the manufacturer's recommendation

  • Optimize based on signal-to-noise ratio in your specific experimental system

For high-sensitivity applications, consider using recombinant monoclonal antibodies like EPR11803-97, which may offer greater consistency between lots compared to polyclonal antibodies .

What approaches can detect different AMOTL1 isoforms or distinguish between AMOTL family members?

For discriminating between closely related AMOTL family proteins or detecting specific AMOTL1 isoforms:

• Selective antibody targeting:

  • Choose antibodies raised against unique regions not conserved between AMOTL1, AMOT, and AMOTL2

  • N-terminal directed antibodies may help distinguish AMOTL1 from other family members

  • For isoform-specific detection, select antibodies targeting regions present in some but not all isoforms

• Western blot optimization:

  • Use gradient gels (4-12%) for better separation of high molecular weight proteins

  • Extended run times can improve separation between AMOTL1 (107 kDa) and AMOTL2

  • Include positive controls expressing single family members for comparison

• qRT-PCR validation:

  • Design primers specific to unique regions of each family member

  • Verify differential expression at mRNA level to complement protein detection

  • Use this approach to correlate with antibody-based detection results

• RNA interference specificity:

  • Design siRNAs specific to each family member

  • Confirm knockdown specificity by western blot using antibodies against all three family members

  • This approach can help validate antibody specificity while also investigating functional redundancy

Research indicates that in gastric cancer cell lines, AMOT is barely expressed, while AMOTL1 and AMOTL2 show relatively high expression levels, emphasizing the importance of discriminating between family members in cancer studies .

How can AMOTL1 antibodies be employed to study its role in developmental disorders and brain function?

Recent research has identified AMOTL1 mutations associated with orofacial clefting syndrome and altered brain development. To investigate these roles:

• Tissue-specific expression profiling:

  • Perform immunohistochemistry on developmental tissue sections using optimized AMOTL1 antibodies (1:50-1:100 dilution)

  • Focus on craniofacial tissues, heart, and brain regions during critical developmental windows

  • Compare normal versus pathological samples (when available)

• Brain-specific analyses:

  • Use IHC or IF to map AMOTL1 expression in different brain regions

  • Correlate with neuroanatomical features observed in Amotl1-depleted mouse brains (enlarged lateral ventricles, cerebral cortex)

  • Implement co-staining with neural cell type markers to identify cell populations expressing AMOTL1

• Mutation impact studies:

  • Generate cell models expressing AMOTL1 variants affecting amino acids 157-161

  • Use antibodies to assess protein stability, localization, and interaction with binding partners

  • Compare wild-type versus mutant protein function in relevant signaling pathways

• Metabolomic correlation:

  • Combine antibody-based detection of AMOTL1 expression with brain metabolite analyses

  • Focus on N-Acetylaspartylglutamic acid (NAAG)/N-Acetyl Aspartate (NAA) ratio and Glutamine (Gln) levels, which were altered in Amotl1-depleted brains

  • This approach can help connect AMOTL1 dysfunction to neurotransmitter metabolism

Research in Amotl1 mutant mice has revealed behavioral phenotypes including hyperactivity, reduced anxiety, altered social behavior, and episodes of backward walking and increased circling behavior, potentially modeling aspects of human neuropsychiatric disorders .

What methodological approaches can detect AMOTL1 post-translational modifications?

To investigate AMOTL1 regulation through post-translational modifications:

• Phosphorylation studies:

  • Immunoprecipitate AMOTL1 using validated antibodies

  • Perform western blot using phospho-specific antibodies or phospho-staining

  • Alternatively, use mass spectrometry to identify phosphorylation sites

  • Compare phosphorylation status under different cellular conditions (e.g., growth factor stimulation, cell density)

• Ubiquitination detection:

  • Transfect cells with HA-tagged ubiquitin

  • Treat with proteasome inhibitor MG132

  • Immunoprecipitate AMOTL1

  • Detect ubiquitination by western blot using anti-HA antibody

  • This approach has been used to demonstrate how YAP1 prevents AMOTL1 from ubiquitin-mediated degradation

• Protein stability assessment:

  • Perform cyclohexamide chase assays

  • Monitor AMOTL1 degradation kinetics under different conditions

  • Research has shown that YAP1 can extend AMOTL1 half-life by preventing its degradation

• Subcellular localization changes:

  • Use cell fractionation followed by western blot or immunofluorescence

  • Monitor AMOTL1 localization changes in response to cell density, mechanical stress, or other stimuli

  • These approaches can reveal regulation through compartmentalization

Research indicates complex regulatory mechanisms where AMOTL1 and YAP1 reciprocally protect each other from degradation through the ubiquitin-proteasome system, suggesting that post-translational modifications significantly influence AMOTL1 function in signaling pathways .

How can I optimize AMOTL1 detection in tissue samples with low expression levels?

When working with tissues where AMOTL1 expression is limited, implement these sensitivity-enhancing techniques:

• Signal amplification methods:

  • Consider using tyramide signal amplification (TSA) for immunohistochemistry

  • Employ biotin-streptavidin systems in conjunction with AMOTL1 antibodies

  • Use ELISA-based detection systems with sensitivity in the 0.078 ng/mL range

• Sample preparation optimization:

  • Test different antigen retrieval methods (citrate buffer, pH 6.0 with pressure cooking has been successful)

  • Optimize fixation protocols to preserve epitope accessibility

  • Consider fresh-frozen samples if formalin fixation compromises antibody binding

• Antibody selection and concentration:

  • Use highly sensitive monoclonal antibodies when available

  • Increase antibody concentration while monitoring background signals

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

• Detection system enhancement:

  • Implement fluorescence-based detection with sensitive cameras for IF

  • For colorimetric IHC, optimize DAB development times and consider alternate chromogens

  • Use enhanced chemiluminescence (ECL) kits with extended exposure times for western blots

Published protocols have successfully detected AMOTL1 in various tissue samples using techniques such as immunohistochemistry with DAB visualization and hematoxylin counterstaining .

What approaches can resolve contradictory AMOTL1 antibody results?

When facing inconsistent results between different AMOTL1 antibodies or detection methods:

• Epitope mapping analysis:

  • Compare the immunogens/epitopes targeted by different antibodies

  • Antibodies targeting different regions of AMOTL1 (N-terminal, internal region, C-terminal) may give different results due to epitope masking, protein processing, or isoform specificity

• Multiple detection technique validation:

  • Confirm protein expression using complementary methods (western blot, IHC, IF)

  • Validate at the mRNA level using qRT-PCR or RNA-seq

  • Consider RNA immunoprecipitation (RIP) to study RNA-protein interactions when relevant

• Experimental condition standardization:

  • Control cell density, passage number, and culture conditions

  • Standardize lysis buffers and protein extraction protocols

  • Document lot numbers of antibodies and reagents

• Functional validation:

  • Implement genetic approaches (siRNA, CRISPR) to validate specificity

  • Perform rescue experiments to confirm phenotype specificity

  • Test antibody reactivity in both overexpression and knockdown systems

• Species-specific considerations:

  • Check sequence homology when working with non-human models

  • Some reported antibodies show variable reactivity across species:

    • Human (100%), Mouse (100%), Cow (93%), Dog (79%), Guinea Pig (100%)

Remember that AMOTL1 function can be context-dependent, with different subcellular localizations (cytosolic in mesenchymal cells, tight junctions and adherens junctions in polarized epithelia) .

How might AMOTL1 antibodies contribute to therapeutic development in cancer research?

Based on recent findings, AMOTL1-targeted approaches offer promising avenues for therapeutic development:

• Biomarker potential:

  • AMOTL1 overexpression correlates with poor prognosis in gastric cancer patients

  • Combined assessment of AMOTL1, nuclear YAP1, and CTGF ("deactivated Hippo" signature) indicates unfavorable clinical outcomes

  • AMOTL1 antibodies could be developed for prognostic immunohistochemistry panels

• Therapeutic target validation:

  • AMOTL1 knockdown enhances the efficacy of first-line anticancer drugs

  • Targeting the AMOTL1-YAP1-CTGF axis could overcome treatment resistance

  • Use antibodies to monitor pathway inhibition during drug development

• Combination therapy assessment:

  • AMOTL1 antibodies can monitor the effects of YAP-TEAD inhibitors like verteporfin (VP)

  • High VP concentrations also cause reduction of AMOTL1 and CTGF expression

  • This approach could help optimize dosing and timing of combination therapies

• Circulating AMOTL1 detection:

  • Develop sensitive ELISA methods to detect AMOTL1 in serum or plasma

  • Current ELISA systems offer sensitivity of 0.078 ng/mL and assay ranges of 0.156-10 ng/mL

  • This could enable non-invasive monitoring of disease progression or treatment response

Recent research demonstrates that AMOTL1 knockout in xenograft models significantly reduces tumor size and weight, while decreasing expression of proliferation markers and increasing apoptotic markers, supporting its potential as a therapeutic target .

How can AMOTL1 antibodies advance our understanding of developmental disorders?

Recent discoveries linking AMOTL1 mutations to a novel orofacial clefting syndrome open new research directions:

• Genotype-phenotype correlation studies:

  • Use antibodies to assess how different AMOTL1 variants (particularly those affecting amino acids 157-161) impact protein expression, stability, and localization

  • Compare findings with patient phenotypes to establish mechanistic connections

  • Develop immunohistochemistry protocols for patient-derived samples

• Developmental pathway analysis:

  • Investigate how AMOTL1 mutations affect critical developmental signaling (Wnt/β-catenin, Hippo)

  • Use antibodies to track AMOTL1 expression patterns during embryonic development

  • Correlate with formation of craniofacial structures, heart, and brain

• Animal model validation:

  • Generate mouse models with AMOTL1 mutations matching human variants

  • Use antibodies to confirm protein expression patterns and levels

  • Correlate with developmental and behavioral phenotypes

• Tissue-specific effects:

  • Implement spatial transcriptomics combined with immunohistochemistry

  • Map AMOTL1 expression across tissues affected in the syndrome (craniofacial, cardiac, auricular, gastrointestinal)

  • Identify cell types where AMOTL1 dysfunction is most impactful

The recent identification of heterozygous missense variants in AMOTL1 defining a new orofacial clefting syndrome highlights the importance of understanding this protein's role in human development and disease .