PDE1A Antibody

What is PDE1A and what cellular processes does it regulate?

PDE1A is a calcium/calmodulin-activated phosphodiesterase enzyme that regulates cyclic nucleotide degradation, specifically cAMP and cGMP. As a member of the PDE1 family, PDE1A serves as a critical intersection point between calcium signaling and cyclic nucleotide pathways. The enzyme is differentially expressed across various cell types including vascular smooth muscle cells, renal tubular epithelial cells, podocytes, and mesangial cells . PDE1A functions as a key regulator in multiple cellular processes including migration, adhesion, and cellular motility, which are critical parameters in cancer cell dissemination . Additionally, recent studies have shown PDE1A's involvement in tumor angiogenesis and metastatic potential, particularly in non-small cell lung cancer (NSCLC) .

What criteria should I consider when selecting a PDE1A antibody for my research?

When selecting a PDE1A antibody for research applications, consider the following critical parameters:

• Antibody specificity: Select antibodies validated to distinguish between PDE1A and other PDE1 isoforms (PDE1B and PDE1C) as these have distinct expression patterns and functions.

• Application compatibility: Ensure the antibody has been validated for your intended application (immunohistochemistry, Western blotting, immunofluorescence, etc.).

• Epitope location: Consider whether the antibody recognizes an epitope that might be masked by protein-protein interactions, such as the YTHDF2 interaction demonstrated in recent studies .

• Cross-reactivity profile: Verify the antibody has been tested against potential cross-reactive proteins, particularly other phosphodiesterases.

• Species reactivity: Confirm compatibility with your experimental model organism (human, mouse, rat, etc.).

For immunofluorescence or proximity ligation assay applications, dilutions around 1:500 have been effective in published research protocols .

How do I validate the specificity of a PDE1A antibody before using it in critical experiments?

Validating PDE1A antibody specificity requires a multi-faceted approach:

• Positive and negative control tissues/cells: Use tissues or cell lines with known high PDE1A expression (such as NSCLC cell lines) as positive controls and compare with low-expressing counterparts (such as normal human lung fibroblasts) .

• siRNA knockdown validation: Transfect cells with PDE1A-specific siRNA and confirm reduction in antibody signal compared to control siRNA.

• Overexpression validation: Compare antibody signal in cells transfected with PDE1A expression vector versus empty vector controls .

• Western blot analysis: Confirm a single band at the expected molecular weight (~61 kDa for human PDE1A).

• Peptide competition assay: Pre-incubate the antibody with a blocking peptide containing the target epitope and confirm signal elimination.

• Multi-antibody validation: Compare staining patterns of different antibodies targeting distinct PDE1A epitopes.

How can I effectively measure PDE1A activity in tissue samples rather than just protein expression?

Measuring PDE1A enzyme activity requires techniques that go beyond detecting protein presence:

• Cyclic nucleotide hydrolysis assay: Quantify cAMP/cGMP degradation rates in tissue lysates with and without calcium/calmodulin stimulation. The calcium/calmodulin-dependent component represents PDE1 activity.

• Selective inhibitor approach: Measure total PDE activity, then repeat with selective PDE1 inhibitors (such as vinpocetine or ITI-214) . The difference represents PDE1 contribution.

• Immunoprecipitation-coupled activity assay: Immunoprecipitate PDE1A using validated antibodies, then measure PDE activity in the precipitate.

• Real-time cGMP monitoring: Use highly sensitive fluorescent indicators to measure dynamic changes in cGMP levels in living cells, as demonstrated in cardiomyocyte studies .

• Differential isoform inhibition: Compare activity profiles after selective knockdown of PDE1A versus PDE1B or PDE1C to distinguish isoform-specific contributions.

For accurate quantification, always include appropriate controls including heat-inactivated samples and samples with EGTA to chelate calcium and inhibit PDE1A activation.

What experimental approaches can resolve contradictory data about PDE1A's role in cell signaling pathways?

When encountering contradictory data regarding PDE1A's signaling role, implement these resolution strategies:

• Cell-type specific analysis: PDE1A functions may vary significantly between cell types. For example, in cardiomyocytes, PDE1 inhibition affects cGMP signaling , while in NSCLC cells, it influences the STAT3 pathway .

• Isoform-specific manipulation: Use not only pharmacological inhibitors but also genetic approaches (siRNA, CRISPR) targeting specific PDE1 isoforms to distinguish their individual contributions.

• Pathway intersection mapping: Since PDE1A sits at the intersection of calcium and cyclic nucleotide signaling, use calcium chelators and pathway-specific inhibitors to delineate which pathway predominates in your system.

• Subcellular localization studies: Determine if PDE1A has different functions in distinct subcellular compartments using fractionation techniques combined with immunofluorescence.

• Temporal dynamics analysis: Implement time-course experiments to resolve seemingly contradictory effects that may occur at different time points after stimulation.

• Protein complex identification: Use proximity ligation assays or co-immunoprecipitation to identify specific protein interactions that may explain context-dependent functions .

How can I distinguish between the roles of different PDE1 isoforms (PDE1A, PDE1B, PDE1C) in my experimental system?

Distinguishing between PDE1 isoforms requires a combinatorial approach:

• Isoform-specific knockdown: Use targeted siRNA for each isoform and compare phenotypic consequences. Research has shown that siPDE1A exerts stronger suppression effects on migration of NCI-H1299 cells than siPDE1B or siPDE1C .

• Expression profiling: Quantify relative expression levels of each isoform in your system using qRT-PCR and Western blotting with isoform-specific antibodies.

• Rescue experiments: After knockdown of all PDE1 activity, selectively re-express individual isoforms to identify their specific contributions.

• Subcellular distribution analysis: Map the distinct subcellular localization patterns of each isoform using isoform-specific antibodies in immunofluorescence studies.

• Calcium sensitivity profiling: The three isoforms have different sensitivities to calcium/calmodulin activation, which can be exploited experimentally.

• Substrate preference analysis: PDE1 isoforms show differential preferences for cAMP versus cGMP, which can be measured in enzymatic assays.

Table adapted from functional studies comparing PDE1 isoforms in NSCLC cells

What are the optimal fixation and antigen retrieval methods for PDE1A immunohistochemistry in different tissue types?

The choice of fixation and antigen retrieval methods significantly impacts PDE1A antibody performance:

• Fixation protocol:

  • For most tissues including lung and kidney, 4% paraformaldehyde (PFA) for 24 hours provides optimal preservation of PDE1A epitopes.

  • For vascular tissues, a shorter fixation (12-16 hours) may yield better results.

  • Fresh-frozen sections may be preferable for certain epitopes sensitive to fixation.

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) at 95-98°C for 20 minutes generally works well for PDE1A.

  • For formalin-fixed tissues, consider Tris-EDTA buffer (pH 9.0) if citrate buffer yields weak signals.

  • Enzymatic retrieval (using proteinase K) is typically not recommended as it may destroy PDE1A epitopes.

• Tissue-specific considerations:

  • Lung tissue: Extended rinsing steps help reduce background from endogenous peroxidases.

  • Brain tissue: Lighter fixation (2% PFA) may improve antibody penetration.

  • Kidney tissue: Requires thorough blocking of endogenous biotin to prevent non-specific staining.

The Human Protein Atlas protocols have successfully visualized PDE1A in normal lung and lung cancer tissues .

How can I optimize proximity ligation assays (PLA) to investigate PDE1A interactions with partner proteins?

Optimizing PLA for PDE1A interaction studies requires careful consideration of several parameters:

• Antibody selection: Use antibodies from different species for PDE1A and its potential interaction partners. For example, rabbit anti-PDE1A combined with mouse antibodies against potential partners like YTHDF2 .

• Antibody validation: Validate both antibodies independently by immunofluorescence before attempting PLA.

• Dilution optimization: Start with 1:500 dilutions for both antibodies as used in previous PLA studies for cGMP-interacting proteins , then optimize as needed.

• Controls:

  • Negative controls: Omit one primary antibody

  • Biological controls: Use cells with PDE1A knockdown

  • Positive controls: Include known protein interaction pairs

• Signal quantification: Use appropriate software to quantify PLA puncta, applying statistical methods such as Kruskal-Wallis for comparing PLA signals between different experimental conditions .

• Protocol refinements:

  • Extend blocking time to 1-2 hours to reduce non-specific interactions

  • Optimize incubation temperature (4°C overnight often yields better results than room temperature)

  • Consider using amplification steps for low-abundance interactions

What strategies can improve detection of phosphorylated states of PDE1A in complex samples?

Detecting phosphorylated PDE1A requires specialized approaches:

• Phospho-specific antibodies: Utilize antibodies specifically targeting known phosphorylation sites of PDE1A. If commercial phospho-specific antibodies are unavailable, consider custom antibody development.

• Phosphatase inhibitors: Include comprehensive phosphatase inhibitor cocktails in all extraction buffers (containing sodium fluoride, sodium orthovanadate, β-glycerophosphate, and phosphatase inhibitor cocktails).

• Enrichment strategies:

  • Phosphoprotein enrichment using TiO₂ or IMAC columns prior to immunoblotting

  • Immunoprecipitation of total PDE1A followed by phospho-specific detection

  • Use of Phos-tag™ SDS-PAGE for mobility shift detection of phosphorylated forms

• Mass spectrometry approaches:

  • Targeted LC-MS/MS after immunoprecipitation

  • Parallel reaction monitoring (PRM) for specific phosphopeptides

  • SILAC or TMT labeling for quantitative phosphoproteomics

• Validation methods:

  • Use phosphatase treatment controls to confirm phospho-specific signals

  • Apply kinase inhibitors relevant to known PDE1A kinases

  • Implement site-directed mutagenesis of phosphorylation sites

How should I design experiments to determine if PDE1A contributes to metastatic potential in my cancer model?

Based on recent findings in NSCLC , a comprehensive experimental design should include:

• Expression correlation analysis:

  • Compare PDE1A expression between primary tumors and matched metastatic lesions

  • Analyze correlation between PDE1A expression and metastasis-free survival in patient cohorts

  • Examine PDE1A expression in cell lines with differential metastatic potential

• In vitro functional assays:

  • Wound healing assays comparing control vs. PDE1A-knockdown/overexpressing cells

  • Transwell migration and invasion assays

  • 3D spheroid invasion assays in appropriate matrix

  • EMT marker assessment (E-cadherin, N-cadherin) by immunoblotting and immunofluorescence

• Mechanistic investigations:

  • Assess PDE1A regulation of specific pathways implicated in metastasis (STAT3, IL-6/JAK)

  • Investigate protein-protein interactions (e.g., PDE1A-YTHDF2) using co-immunoprecipitation and PLA

  • Examine effects on mRNA stability of metastasis-related genes

• In vivo metastasis models:

  • Tail vein injection metastasis model with PDE1A-modulated cells

  • Orthotopic implantation with spontaneous metastasis assessment

  • Analyze metastatic nodules in lungs or other organs

• Pharmacological validation:

  • Test effects of selective PDE1 inhibitors like vinpocetine on metastatic potential

  • Compare with genetic manipulation to confirm target specificity

What controls are essential when investigating the effects of PDE1A inhibition on cGMP/cAMP-dependent pathways?

When studying PDE1A inhibition effects on cyclic nucleotide signaling, incorporate these essential controls:

• Inhibitor specificity controls:

  • Compare multiple structurally distinct PDE1 inhibitors (vinpocetine, ITI-214, LY1)

  • Include PDE1A genetic knockdown alongside pharmacological inhibition

  • Use inactive structural analogs of inhibitors where available

• Pathway validation controls:

  • Include direct activators of adenylyl cyclase (forskolin) or guanylyl cyclase (NO donors)

  • Use cell-permeable cAMP/cGMP analogs to confirm downstream effects

  • Apply specific inhibitors of PKA (e.g., H-89) or PKG (e.g., KT5823) to distinguish pathway contributions

• Temporal controls:

  • Perform detailed time-course experiments to capture both rapid and delayed responses

  • Implement washout experiments to confirm reversibility

• Readout validation:

  • Directly measure cAMP/cGMP levels using ELISA or real-time fluorescent sensors

  • Monitor phosphorylation of known PKA substrates (CREB, RyR) and PKG substrates (VASP)

  • Examine calcium-dependent effects with and without calcium chelators

• Cell type controls:

  • Compare effects in cells with high versus low endogenous PDE1A expression

  • Test in multiple cell types relevant to the biological question

These controls will help distinguish direct effects of PDE1A inhibition from off-target effects and determine pathway specificity.

How can I effectively study the interaction between PDE1A and YTHDF2 in regulating the STAT3 pathway?

Recent research has identified a novel PDE1A interaction with YTHDF2 that affects STAT3 signaling in NSCLC . To investigate this interaction effectively:

• Interaction validation approaches:

  • Co-immunoprecipitation with antibodies against both PDE1A and YTHDF2

  • Proximity ligation assay to visualize interactions in situ

  • FRET or BiFC assays for dynamic interaction studies in living cells

  • Domain mapping using truncated constructs to identify interaction regions

• Functional relationship studies:

  • Knockdown YTHDF2 and assess effects on PDE1A function and localization

  • Knockdown PDE1A and examine YTHDF2's RNA-binding activity

  • Perform rescue experiments with wild-type and mutant constructs

• m6A-mediated regulation analysis:

  • Perform MeRIP-seq to identify m6A-modified transcripts affected by PDE1A-YTHDF2 interaction

  • RNA stability assays for candidate targets like SOCS2

  • Use m6A writer (METTL3/14) or eraser (FTO, ALKBH5) manipulations to modify the pathway

• STAT3 pathway investigation:

  • Monitor STAT3 phosphorylation status with PDE1A/YTHDF2 manipulation

  • Assess nuclear translocation of STAT3 using fractionation and imaging

  • Measure STAT3 target gene expression

  • Test IL-6 stimulation with and without PDE1A/YTHDF2 modulation

• Integrated multi-omics approach:

  • Combine transcriptomics, proteomics, and phosphoproteomics data

  • Integrate with publicly available datasets on YTHDF2-interacting RNAs

  • Perform GSEA and ORA analyses similar to those used in previous studies

How can PDE1A antibodies be used to stratify cancer patients for potential PDE1 inhibitor therapy?

PDE1A antibodies can be valuable tools for patient stratification in precision oncology:

• Tissue microarray analysis:

  • Develop immunohistochemistry scoring systems for PDE1A expression

  • Correlate expression with patient outcomes and treatment responses

  • Create cutoff values for high versus low expression groups

• Multimarker panels:

  • Combine PDE1A staining with other markers in the pathway (YTHDF2, STAT3, SOCS2)

  • Develop integrated scoring systems that predict response to PDE1 inhibition

  • Validate in retrospective patient cohorts before prospective studies

• Liquid biopsy applications:

  • Explore detection of PDE1A in circulating tumor cells

  • Investigate PDE1A in exosomes as potential biomarkers

  • Correlate with tissue expression and clinical outcomes

• Functional testing:

  • Develop ex vivo drug sensitivity assays using patient-derived tumor samples

  • Correlate PDE1A expression with sensitivity to PDE1 inhibitors

What methodological approaches can distinguish between direct and indirect effects of PDE1A on cardiovascular function?

PDE1A's role in cardiovascular physiology requires careful experimental design to separate direct from indirect effects:

• Cell-type specific analyses:

  • Use cardiomyocyte-specific conditional PDE1A knockout models

  • Compare with vascular smooth muscle-specific knockouts

  • Analyze endothelial-specific effects separately

• Ex vivo preparations:

  • Isolated heart preparations to evaluate direct cardiac effects

  • Isolated vessel myography to assess direct vascular effects

  • Compare responses in tissue from knockout versus wild-type animals

• Pharmacological dissection:

  • Use selective PDE1 inhibitors with different pharmacokinetic profiles

  • Apply inhibitors before and after autonomic blockade

  • Compare acute versus chronic inhibition effects

• Signaling pathway resolution:

  • Monitor real-time cGMP dynamics in cardiomyocytes using fluorescent indicators

  • Determine PKG-dependent versus PKA-dependent effects

  • Assess calcium handling parameters independently

• Hemodynamic separations:

  • Measure blood pressure effects in normotensive versus hypertensive rats

  • Distinguish between cardiac output effects and vascular resistance changes

  • Analyze pressure-volume relationships to separate preload, afterload, and contractility effects

PDE1 inhibition has been shown to reduce blood pressure in both normotensive and spontaneously hypertensive rats, suggesting direct vascular effects .

How can antibody-based approaches help elucidate the role of PDE1A in kidney disease pathophysiology?

Antibody-based techniques can provide valuable insights into PDE1A's role in kidney disease:

• Cell-type mapping:

  • Perform high-resolution co-localization studies to map PDE1A expression in specific nephron segments

  • Use dual-immunostaining with nephron segment markers (podocin, AQP1, NKCC2, NCC)

  • Compare expression patterns in healthy versus diseased kidneys

• Pathological correlations:

  • Quantify PDE1A expression in kidney biopsies from various nephropathies

  • Correlate with markers of kidney injury (KIM-1, NGAL)

  • Assess relationship with fibrosis markers and inflammatory infiltrates

• Mechanistic studies:

  • Use phospho-specific antibodies to track PDE1A activation status

  • Perform PLA to identify disease-specific protein interaction networks

  • Combine with functional readouts of cAMP/cGMP signaling

• Intervention monitoring:

  • Track changes in PDE1A expression during progression of diabetic kidney disease

  • Monitor effects of PDE1 inhibitors on albuminuria and kidney function

  • Evaluate changes in PDE1A activation with different therapeutic approaches

Research has shown that PDE1 inhibition with selective inhibitors like LY1 can significantly improve renal function in diabetic kidney disease models, reducing albuminuria , suggesting antibody-based monitoring of PDE1A could help track disease progression and treatment response.