ATP9A Antibody

What is ATP9A and what cellular functions does it serve?

ATP9A is a lipid flippase of the class II P4-ATPases involved in cellular vesicle trafficking. It plays critical roles in:

• Endosomal recycling pathway through modulation of small GTPases RAB5 and RAB11 activation

• Recycling of transferrin and glucose transporter 1 from endosomes to the plasma membrane

• Maintenance of neurite morphology and synaptic transmission

ATP9A catalyzes the reaction: ATP + H₂O + phospholipid(In) = ADP + phosphate + phospholipid(Out), although its flippase activity towards membrane lipids and its role in membrane asymmetry remains to be definitively proven .

What is the subcellular localization of ATP9A?

ATP9A exhibits specific subcellular localization patterns:

• Localizes to early/recycling endosomes and the trans-Golgi network (TGN)

• Colocalizes with EEA1 (early endosome marker), transferrin receptor (TfnR, early/recycling endosome marker), and TGN46 (TGN marker)

• Does not colocalize with Lamp-1 (late endosome marker) or GM130 (cis-Golgi marker)

• Specifically localizes to phosphatidylserine-positive early and recycling endosomes

When using immunofluorescence to detect ATP9A, optimal results are achieved by using markers for early/recycling endosomes for colocalization studies.

What applications are ATP9A antibodies validated for?

ATP9A antibodies have been validated for multiple applications:

For optimal results, antibody dilutions should be titrated for each specific application and sample type.

What is known about ATP9A expression across different tissues?

ATP9A shows variable expression across tissues:

• High expression in brain tissue, particularly in the cortex and CA3 region of the hippocampus

• Detectable expression in liver, spleen, and kidney

• Expression in leukocytes from human blood

• Available in cell lines including HeLa, MCF7, and HepG2

In situ hybridization studies confirm that ATP9A is abundant in the six layers of the cortex and in the CA3 region of the hippocampus in wild-type mice .

How do molecular interactions between ATP9A and ATP9B affect experimental design?

ATP9A forms both homomeric and heteromeric complexes with ATP9B, which impacts experimental approaches:

• Co-immunoprecipitation analysis confirms that ATP9A and ATP9B interact specifically, while neither interacts with ATP11C

• Blue native PAGE (BN-PAGE) reveals that ATP9A-HA migrates at approximately 470-480 kDa, while ATP9B-HA migrates at approximately 240 kDa

• ATP9B contributes to the localization of ATP9A to the Golgi complex; in ATP9B-KO cells, ATP9A shows increased endosomal localization

When designing experiments to study ATP9A, researchers should consider:

• Using ATP9A/9B double knockout cell lines to avoid interference from endogenous proteins

• Examining both homomeric and heteromeric complexes through co-immunoprecipitation and native PAGE

• Investigating the localization of ATP9A in the presence and absence of ATP9B

What are the optimal protocols for detecting ATP9A in knockout models?

When working with ATP9A knockout models:

• mRNA detection: RT-qPCR using validated primers can confirm knockout efficiency. In ATP9A knockout mice, very low mRNA levels were detected in brain tissues and subregions (cortex, hippocampus, striatum, midbrain, thalamus) as well as liver, spleen, and kidney .

• Protein detection: Western blot analysis using validated antibodies. Note that due to low specificity of some ATP9A antibodies, a weak normal band (~100 kDa) might be observed even in knockout samples .

• Tissue visualization: In situ hybridization for mRNA detection in tissue sections. This technique confirms ATP9A abundance in cortical layers and hippocampal CA3 region in wild-type animals, with significant reduction in knockout models .

• Behavioral phenotyping: ATP9A knockout mice show decreased muscle strength, memory deficits, and hyperkinetic movement disorder, recapitulating symptoms observed in patients with ATP9A mutations .

How can I investigate ATP9A's role in endosomal recycling pathways?

To study ATP9A's function in endosomal recycling:

• Knockdown experiments: Use siRNAs targeting coding (ATP9A-1) and noncoding (ATP9A-2) regions of ATP9A. Verify knockdown by qRT-PCR and Western blot in cells expressing tagged ATP9A .

• Transferrin recycling assay:

  • Incubate cells with fluorescently-labeled transferrin

  • Allow internalization, then wash to remove surface-bound transferrin

  • Measure recycling kinetics through flow cytometry or microscopy

  • Compare recycling rates between control and ATP9A-depleted cells

• BFA treatment analysis:

  • Treat cells with Brefeldin A (BFA) to induce tubulation of TfnR-positive endosomes

  • Observe ATP9A localization to determine its association with tubulated endosomes

  • Use as a method to confirm endosomal localization of ATP9A

• RAB modulation:

  • Investigate ATP9A's role in modulating RAB5 and RAB11 activation

  • Compare RAB activation states between wild-type and ATP9A-deficient cells

  • Use dominant-negative or constitutively active RAB mutants to place ATP9A in the RAB-dependent trafficking pathway

What are the challenges in detecting endogenous ATP9A protein?

Researchers face several challenges when detecting endogenous ATP9A:

• Antibody specificity issues: Studies note the "low specificity of ATP9A antibodies," with weak bands sometimes visible even in knockout samples .

• Protein size verification: The calculated molecular weight of ATP9A is 119 kDa, but it is often observed at approximately 105 kDa in Western blots .

• Cross-reactivity: Careful validation is needed due to the homology between ATP9A and other P4-ATPase family members.

• Expression levels: Endogenous expression may be low in certain cell types, requiring sensitive detection methods.

Recommended approaches:

• Use multiple antibodies targeting different epitopes

• Include appropriate positive controls (tissues with known high expression)

• Implement knockdown/knockout controls to confirm specificity

• Consider using tagged versions for initial localization studies

How do genetic mutations in ATP9A affect experimental outcomes?

ATP9A mutations have significant impacts that should be considered in experimental design:

• Nonsense mutations: Mutations c.433C>T/c.658C>T/c.983G>A (p.Arg145*/p.Arg220*/p.Trp328*) in ATP9A cause autosomal recessive neurodevelopmental disorders . These mutations generate premature termination codons and cause translational termination of ATP9A .

• Expression effects: In patients with ATP9A mutations, expression is significantly decreased at both mRNA and protein levels .

• Subcellular localization: ATP9A pathogenic mutants show aberrant subcellular localization and cause abnormal endosomal recycling .

• Experimental models: Mouse models with Atp9a deletion show:

  • Decreased survival (median survival time of 252.5 days vs 627 days for wild-type)

  • Complete infertility in homozygous knockout matings

  • Behavioral phenotypes including decreased muscle strength and memory deficits

  • Abnormal neurite morphology and impaired synaptic transmission in primary motor cortex and hippocampus

When studying ATP9A mutations, researchers should consider:

• Effects on both protein expression and localization

• Functional consequences on endosomal recycling pathways

• Impact on interactions with binding partners

• Phenotypic outcomes in cellular and animal models

What are the recommended protocols for immunofluorescence staining of ATP9A?

For optimal ATP9A immunofluorescence staining:

Protocol:

• Cell preparation:

  • Culture cells on glass coverslips to 70-80% confluency

  • Fix with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization and blocking:

  • Permeabilize with 0.1% Triton X-100 in PBS for 10 minutes

  • Block with 3% BSA in PBS for 1 hour at room temperature

• Primary antibody incubation:

  • Dilute ATP9A antibody 1:10-1:100 in blocking solution

  • Incubate overnight at 4°C in a humidified chamber

• Secondary antibody incubation:

  • Wash 3× with PBS

  • Incubate with appropriate fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 488-conjugated anti-rabbit IgG) at 1:500 for 1 hour at room temperature

• Counterstaining and mounting:

  • Wash 3× with PBS

  • Counterstain nuclei with DAPI (1:1000) for 5 minutes

  • Mount with anti-fade mounting medium

Co-staining recommendations:

• For endosomal localization: Co-stain with EEA1 or TfnR

• For TGN localization: Co-stain with TGN46

• Avoid co-staining with Lamp-1 or GM130 as ATP9A does not colocalize with these markers

How can I optimize Western blot detection of ATP9A?

For reliable Western blot detection of ATP9A:

Protocol:

• Sample preparation:

  • Lyse cells in buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, and protease inhibitor mixture

  • Incubate for 30 minutes on ice with occasional vortexing

  • Centrifuge at 15,000×g for 15 minutes at 4°C

  • Collect supernatant and determine protein concentration

• SDS-PAGE:

  • Load 30-50 μg of protein per lane

  • Use 7.5% or 4-12% gradient gels for optimal separation

  • Run gel at 100-120V until dye front reaches bottom

• Transfer:

  • Transfer to PVDF membrane at 100V for 90 minutes or 30V overnight at 4°C

  • Use wet transfer for high molecular weight proteins like ATP9A

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in TBS-T for 1 hour at room temperature

  • Incubate with ATP9A antibody (1:500-1:3000) in blocking buffer overnight at 4°C

  • Wash 3× with TBS-T

  • Incubate with HRP-conjugated secondary antibody (1:5000-1:50000) for 1 hour

  • Wash 4× with TBS-T

• Detection:

  • Develop using ECL substrate

  • Expected band size: calculated 119 kDa, observed approximately 105 kDa

Optimization notes:

• Include positive controls (MCF7 or HepG2 cells)

• Consider using gradient gels for better resolution

• Optimize primary antibody concentration for each application

• Note that weak bands may appear even in knockout samples due to antibody cross-reactivity

What approaches can I use to study ATP9A in neuronal systems?

To investigate ATP9A in neuronal systems:

• Primary neuron cultures:

  • Isolate neurons from mouse cortex or hippocampus (regions with high ATP9A expression)

  • Analyze neurite morphology with phase-contrast microscopy or MAP2/Tau immunostaining

  • Examine ATP9A localization using immunofluorescence

• Synaptic function assessment:

  • Use electrophysiology to measure synaptic transmission

  • ATP9A knockout mice show impaired synaptic transmission in primary motor cortex and hippocampus

• Genetic manipulation approaches:

  • CRISPR/Cas9 system for ATP9A gene inactivation

  • Design guide RNAs targeting early exons

  • Use PX459 vector system for transfection

  • Verify editing by PCR and sequencing of genomic DNA

• Behavioral analysis in mouse models:

  • Coat hanger test and wire hang test for analyzing neuromuscular strength and coordination

  • Y-maze tests to assess spatial working memory

  • Open field test and elevated plus maze for hyperactivity assessment

• Molecular pathway analysis:

  • Investigate RAB5 and RAB11 activity-dependent endosomal recycling pathway

  • ATP9A deficiency leads to inactivation of these RABs in neurons

How can I validate the specificity of ATP9A antibodies?

To ensure ATP9A antibody specificity:

• Knockout/knockdown controls:

  • Use CRISPR/Cas9-generated ATP9A knockout cells

  • Alternatively, perform siRNA knockdown targeting coding (ATP9A-1) or noncoding (ATP9A-2) regions

  • Verify knockdown efficiency by qRT-PCR and Western blot

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Compare staining patterns with and without peptide blocking

  • Specific signals should be abolished by peptide competition

• Multiple antibody validation:

  • Use antibodies targeting different epitopes of ATP9A

  • Compare staining patterns to confirm consistency

  • Available antibodies include rabbit polyclonal (19504-1-AP), mouse monoclonal (3G2), and others

• Orthogonal validation:

  • Correlate protein detection with RNAseq data

  • Some commercial antibodies are validated using orthogonal RNAseq approaches

• Expected molecular weight verification:

  • Calculated molecular weight: 119 kDa

  • Observed molecular weight: approximately 105 kDa

  • Be aware that weak bands at normal size (~100 kDa) might appear even in knockout samples due to antibody specificity issues

What are the best practices for examining ATP9A-protein interactions?

For investigating ATP9A protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Establish stable cell lines expressing differentially tagged versions (HA-tagged and FLAG-tagged ATP9A)

  • Prepare cell lysates using mild detergent buffers to preserve protein-protein interactions

  • Perform immunoprecipitation using anti-tag antibodies

  • Analyze precipitates by Western blot

  • Include appropriate controls (e.g., ATP11C as a negative control)

• Blue native PAGE (BN-PAGE):

  • Use non-denaturing conditions to preserve protein complexes

  • ATP9A typically appears at approximately 470-480 kDa, indicating complex formation

  • Include appropriate controls to distinguish homomeric vs. heteromeric complexes

• Proximity ligation assay (PLA):

  • Allows visualization of protein-protein interactions in situ

  • Particularly useful for detecting endogenous interactions in intact cells

• Mass spectrometry-based approaches:

  • Perform immunoprecipitation followed by mass spectrometry

  • Identify novel interaction partners

  • Validate candidates through orthogonal methods

• Considerations for specific interactions:

  • ATP9A forms complexes with ATP9B but not with ATP11C

  • ATP9A does not interact with CDC50A, unlike other P4-ATPases

  • ATP9A interacts with RAB5 and RAB11, modulating their activation