ATP2C1 Antibody

What is ATP2C1 and what cellular functions does it regulate?

ATP2C1, also known as PMR1, encodes the human secretory pathway Ca²⁺/Mn²⁺-ATPase protein 1 (hSPCA1), which belongs to the family of P-type cation transport ATPases. This magnesium-dependent enzyme catalyzes ATP hydrolysis coupled with calcium and manganese transport into the Golgi complex . ATP2C1 is related to other P-type ATPases, including sarco(endo)plasmic calcium ATPase (SERCA) and plasma membrane calcium ATPase (PMCA). The protein exists as multiple splice variants with slight structural differences . ATP2C1 is crucial for maintaining epidermal integrity by regulating intracellular calcium signaling, and plays roles in oxidative stress regulation, DNA damage response, and post-translational processing of secretory proteins .

What is the expression pattern of ATP2C1 across different tissues?

ATP2C1 is ubiquitously expressed across multiple tissues, including skin, brain, skeletal muscle, placenta, heart, and lungs . Despite this widespread expression, ATP2C1 haploinsufficiency in Hailey-Hailey disease primarily affects keratinocytes, suggesting tissue-specific sensitivity to ATP2C1 dosage . Interestingly, primary human keratinocytes show particularly bright immunofluorescent expression of ATP2C1 compared to other cell types, and this expression is regulated by extracellular calcium levels. When keratinocytes are cultured in high calcium (1.25 mM), ATP2C1 immunofluorescence intensity markedly decreases compared to low calcium (0.09 mM) conditions .

What applications are ATP2C1 antibodies suitable for?

ATP2C1 antibodies are validated for multiple research applications, depending on the specific antibody clone and format:

Some antibodies target specific amino acid regions of ATP2C1, such as AA 119-269, AA 400-660, or AA 491-605, allowing for domain-specific analyses .

What is the subcellular localization of ATP2C1?

ATP2C1 predominantly localizes to the Golgi complex in a calcium-dependent manner . Immunofluorescence studies show ATP2C1 co-localizing with Golgi mannosidase II, confirming its Golgi localization . When cells are treated with brefeldin A (which disrupts the Golgi apparatus), ATP2C1 redistributes to a pattern similar to that of ER-localized protein disulfide isomerase . This localization is consistent with its function in maintaining calcium and manganese homeostasis in the secretory pathway. Interestingly, the detection of endogenous ATP2C1 by immunofluorescence is markedly increased in cells cultured in low calcium media, suggesting that calcium levels influence either the expression or epitope accessibility of ATP2C1 .

How does ATP2C1 deficiency affect the DNA damage response (DDR)?

ATP2C1 deficiency leads to significant dysregulation of the DNA damage response pathway. RNA-seq experiments have revealed that the DDR is consistently downregulated in keratinocytes derived from Hailey-Hailey disease lesions . While oxidative stress typically activates the DDR, ATP2C1 knockdown paradoxically impairs this response. The mechanism involves increased oxidative stress upon ATP2C1 inactivation, which unexpectedly leads to enhanced Notch1 activation . This activated Notch1 significantly reduces both phosphorylated and total ATM (a key DDR protein), compromising the cell's ability to respond to DNA damage. Importantly, inhibiting Notch1 activation with γ-secretase inhibitors (GSI) prevents the reduction in ATM levels in ATP2C1-depleted cells . This suggests a regulatory cascade where ATP2C1 deficiency → increased oxidative stress → Notch1 activation → impaired DDR.

What is the relationship between ATP2C1, Notch1 signaling, and keratinocyte differentiation?

The interaction between ATP2C1 and Notch1 signaling reveals complex regulatory mechanisms affecting keratinocyte biology:

• ATP2C1 deficiency enhances Notch1 expression, which can promote keratinocyte differentiation .

• In normal human skin, Notch1 shows strong staining throughout the epidermal layer, appearing as brown or tan particles or clumps .

• In skin tissues from all examined HHD patients, Notch1 signals are significantly weaker compared to normal controls .

• ATP2C1 RNA interference enhances Notch1 expression while promoting keratinocyte differentiation .

These seemingly contradictory findings (enhanced Notch1 expression with siRNA but decreased expression in patient tissues) suggest complex temporal dynamics in Notch1 regulation during disease progression. The acute loss of ATP2C1 may initially upregulate Notch1, while chronic deficiency in patient tissues eventually leads to downregulation, potentially as a compensatory mechanism.

How does ATP2C1 deficiency impact cellular stress responses and protein processing?

ATP2C1 deficiency causes multiple defects in secretory pathway function and stress responses:

What are the optimal conditions for ATP2C1 antibody use in immunofluorescence studies?

For optimal immunofluorescence detection of ATP2C1:

• Fixation: For cultured cells, 4% paraformaldehyde fixation for 15 minutes at room temperature preserves Golgi structure while maintaining antibody accessibility.

• Permeabilization: 0.1% Triton X-100 for 5 minutes provides sufficient permeabilization without disrupting Golgi morphology.

• Blocking: 3-5% BSA or normal serum (matching the secondary antibody host) for 30-60 minutes reduces background.

• Calcium manipulation: Consider manipulating extracellular calcium levels, as ATP2C1 detection is significantly enhanced in cells cultured in low calcium (0.09 mM) media compared to high calcium (1.25 mM) conditions .

• Co-staining markers: Include Golgi markers (e.g., Golgi mannosidase II) to confirm proper localization .

• Controls: Include positive controls (cells known to express ATP2C1) and negative controls (primary antibody omission or ATP2C1-depleted cells).

• Antibody selection: For detecting endogenous ATP2C1, select antibodies validated for immunofluorescence applications. Some researchers have found that while certain ATP2C1 antibodies may not reliably detect endogenous protein by Western blotting, they can yield strong signals by immunofluorescence .

How can researchers effectively differentiate between ATP2C1 isoforms?

ATP2C1 exists as multiple splice variants that differ by approximately 20 amino acids . To distinguish between these isoforms:

• Isoform-specific antibodies: Select antibodies targeting regions that differ between isoforms. For example, antibodies targeting AA 119-269, AA 400-660, or other specific domains may have different affinities for various isoforms .

• RT-PCR approach: Design primers flanking alternatively spliced regions to amplify and distinguish different mRNA isoforms.

• Western blot resolution: Use lower percentage (6-8%) SDS-PAGE gels with extended run times to better separate high molecular weight ATP2C1 isoforms.

• Immunoprecipitation followed by mass spectrometry: For definitive isoform identification, immunoprecipitate ATP2C1 and analyze by mass spectrometry to identify specific peptides unique to each isoform.

• Recombinant expression controls: Include recombinant expression of known ATP2C1 isoforms as positive controls for size comparison in Western blot analyses.

What controls are essential when using ATP2C1 antibodies in experimental studies?

Proper controls are critical for reliable interpretation of ATP2C1 antibody-based experiments:

• Positive tissue controls: Known ATP2C1-expressing tissues such as skin, which shows particularly strong expression in keratinocytes .

• Negative controls:

  • Primary antibody omission

  • Isotype control antibodies (matching the primary antibody isotype, e.g., IgG1 for monoclonal antibodies like clone 4G12)

  • ATP2C1-depleted cells (via siRNA knockdown)

• Validation across methods: Confirm findings with multiple detection methods (e.g., immunofluorescence, Western blot, qPCR).

• Rescue experiments: For knockdown studies, include rescue with wild-type ATP2C1 to confirm specificity of observed phenotypes.

• Antibody validation: When using new lots of antibodies, validate using known positive controls and Western blot to confirm specificity.

• Cross-reactivity testing: Test reactivity with closely related proteins (other P-type ATPases) to ensure specificity.

What methods can be used to study calcium dynamics in relation to ATP2C1 function?

To investigate calcium dynamics in ATP2C1 research:

• Golgi-targeted calcium sensors: Genetically encoded calcium indicators (GECIs) targeted to the Golgi apparatus provide direct measurement of Golgi calcium levels influenced by ATP2C1 activity.

• Live cell calcium imaging: Calcium-sensitive dyes like Fura-2 or Fluo-4 can be used to monitor cytosolic calcium levels in ATP2C1-deficient cells compared to controls.

• Calcium chelation experiments: BAPTA-AM (cell-permeant calcium chelator) can be used to determine whether phenotypes observed in ATP2C1-deficient cells are calcium-dependent.

• Calcium add-back experiments: Modulating extracellular calcium levels (as done in studies showing calcium-dependent expression of ATP2C1) can reveal calcium-sensitive processes.

• Manganese competition assays: Since ATP2C1 transports both calcium and manganese, competition assays can reveal the relative importance of each ion for specific cellular functions.

• Calcium perturbation agents: Compounds like thapsigargin (SERCA inhibitor) or ionomycin (calcium ionophore) can be used in combination with ATP2C1 manipulation to dissect calcium pathway interactions.

How can researchers address weak or non-specific ATP2C1 antibody signals?

When encountering weak or non-specific signals with ATP2C1 antibodies:

• Application-specific optimization: Some ATP2C1 antibodies may work well for immunofluorescence but poorly for Western blotting, as observed with antibody 161, which yielded strong immunofluorescence signals but could not reliably detect endogenous ATP2C1 by Western blotting despite purification .

• Antigen retrieval for IHC/IF: For formalin-fixed tissues, optimize antigen retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0 or EDTA buffer pH 9.0).

• Signal amplification systems: Consider using tyramide signal amplification (TSA) or other amplification methods for weak signals.

• Calcium modulation: ATP2C1 detection by immunofluorescence is significantly enhanced in cells cultured in low calcium media , suggesting that experimental conditions may dramatically affect epitope accessibility.

• Protein enrichment: For Western blotting, consider Golgi membrane enrichment protocols to concentrate ATP2C1 before analysis.

• Alternative antibody clones: If possible, test multiple antibody clones targeting different epitopes, such as monoclonal 4G12 versus 2G1 .

• Recombinant positive controls: Include lysates from cells overexpressing ATP2C1 as positive controls to confirm the expected band size.

What approach should be used to study ATP2C1 in disease models?

For investigating ATP2C1 in disease contexts, particularly Hailey-Hailey disease:

• Patient-derived keratinocytes: Primary keratinocytes isolated from HHD patient lesions provide the most clinically relevant model for studying ATP2C1 deficiency .

• Genetic analysis: PCR amplification and sequencing of all ATP2C1 exons from patient genomic DNA can identify novel disease-causing mutations .

• Protein expression analysis: Combine immunostaining techniques to examine the expression pattern of multiple proteins in the same tissues, including:

  • hSPCA1 (ATP2C1 protein product)

  • miR-203 (a microRNA potentially involved in regulation)

  • p63 (epidermal transcription factor)

  • Notch1 (signaling protein affected by ATP2C1 deficiency)

  • HKII (hexokinase II, related to energy metabolism)

• Comparative analysis: Always include matched normal skin controls when analyzing HHD patient samples, as Notch1 staining has been shown to be weaker in HHD patient skin compared to normal controls .

• Integrated approach: Combine genetic, protein expression, and functional studies to establish genotype-phenotype correlations in HHD patients with different ATP2C1 mutations.

What emerging applications of ATP2C1 antibodies show promise for research advancement?

Several emerging research areas could benefit from advanced ATP2C1 antibody applications:

• Single-cell analysis: Combining ATP2C1 antibodies with single-cell technologies could reveal cell-specific expression patterns and heterogeneity in normal and diseased tissues.

• Proximity labeling approaches: BioID or APEX2 fusions with ATP2C1 could identify novel interacting partners in the Golgi environment.

• Super-resolution microscopy: Advanced imaging techniques could better resolve the precise subcompartmental localization of ATP2C1 within the Golgi complex.

• Therapeutic antibody development: Given ATP2C1's role in Hailey-Hailey disease, therapeutic antibodies or antibody-drug conjugates targeting surface proteins on affected keratinocytes could offer novel treatment approaches.

• Cancer research applications: Since heterozygous ATP2C1 knockout mice show susceptibility to squamous cell tumors , ATP2C1 antibodies may have applications in cancer research and diagnostics.