pald1 Antibody

What is PALD1 and what are its key biological functions?

PALD1, also known as Paladin or KIAA1274, functions as a phosphoinositide phosphatase that regulates endosomal trafficking. It plays a critical role in vascular biology, particularly as a negative regulator of endothelial proliferation and angiogenic sprouting . PALD1 is expressed predominantly in endothelial cells, in mural cells of certain larger vessels, potentially in microglia within the brain, and in peripheral blood mononuclear cells .

At the molecular level, PALD1 regulates VEGFR2 internalization and signaling. Deficiency in PALD1 leads to faster VEGFR2 internalization to EEA1+ endosomes and increased phosphorylation of ERK1/2 following VEGF-A stimulation, suggesting its role in modulating growth factor signaling pathways . Diseases associated with PALD1 include Alzheimer Disease 7 and Otomycosis, highlighting its potential significance in both neurodegenerative and infectious conditions .

What applications are PALD1 antibodies validated for and what are the recommended dilutions?

PALD1 antibodies have been validated for multiple experimental applications with specific dilution ranges that vary by application and antibody source:

For optimal results, researchers should first validate these dilutions in their specific experimental systems, as factors such as expression levels, sample type, and detection methods can influence antibody performance.

What is the expected molecular weight of PALD1 in Western blotting?

When conducting Western blot analysis, researchers should be aware that while the calculated molecular weight of PALD1 is approximately 151 kDa (1383 amino acids), the observed molecular weight typically presents as bands at 95 kDa and 140 kDa . This discrepancy between calculated and observed weights is not uncommon in protein research and may reflect:

• Post-translational modifications affecting protein mobility

• Presence of multiple isoforms or splice variants

• Proteolytic processing during sample preparation

For validation purposes, researchers should expect to observe these characteristic bands, and the absence of either band might indicate issues with antibody specificity, sample preparation, or protein expression levels in their experimental system.

What are the recommended specimen preparation methods for PALD1 immunohistochemistry?

For optimal detection of PALD1 in tissue sections, researchers should consider the following preparation methods:

• Fixation: Standard formalin fixation is generally compatible with PALD1 detection.

• Antigen retrieval: TE buffer at pH 9.0 is recommended, although citrate buffer at pH 6.0 can also be used as an alternative .

• Blocking: Use of buffers containing BSA, FCS, and mild detergents (e.g., 0.2% Tween 20/3% BSA/5% FCS/0.05% Sodium Deoxycholate in PBS) has been successfully applied for PALD1 immunostaining .

• Primary antibody incubation: Typically performed overnight at 4°C at dilutions between 1:50-1:500 .

• Detection systems: Compatible with standard secondary antibody detection methodologies.

Positive IHC has been successfully demonstrated in human tissues including colon cancer, breast cancer, normal colon tissue, and ovary tumor tissue , providing useful positive controls for researchers.

What is the cellular localization of PALD1?

PALD1 primarily exhibits cytoplasmic localization . This can be verified through immunofluorescence staining with specific anti-PALD1 antibodies. For optimal visualization, researchers have successfully used protocols involving:

• Cell fixation with 3% paraformaldehyde

• Permeabilization with 0.1% Triton X-100

• Primary antibody incubation (e.g., rabbit anti-paladin, 1:200, Atlas Antibodies, HPA015696) overnight at 4°C

• Detection with fluorophore-conjugated secondary antibodies

• Nuclear counterstaining with DAPI

This methodology allows visualization of the cytoplasmic distribution pattern of PALD1 and can be combined with markers for specific subcellular structures to further characterize its precise localization within the cytoplasmic compartment.

How does PALD1 regulate VEGFR2 trafficking and signaling in endothelial cells?

PALD1 functions as a critical regulator of VEGFR2 internalization and early endosomal trafficking in endothelial cells. Research utilizing PALD1 knockdown models has revealed several key mechanisms:

• PALD1 deficiency results in a marked increase (35-51%) in the total basal VEGFR2 pool, suggesting a role in regulating steady-state receptor levels .

• Following VEGF-A stimulation, PALD1-silenced cells demonstrate significantly faster VEGFR2 internalization to early endosomes. After 15 minutes of VEGF-A treatment, the internalized VEGFR2 pool in PALD1-silenced endothelial cells was nearly twice that of control cultures .

• PALD1 knockdown cells exhibit increased numbers of EEA1+ vesicles and EEA1/VEGFR2 double-positive structures after just 2 minutes of VEGF-A stimulation, with further increases at 10 minutes .

• PALD1 appears to regulate phosphoinositide metabolism, as PALD1 knockdown results in a prominent increase of vesicular PI(4,5)P2 signal after VEGF-A stimulation .

• The altered trafficking correlates with enhanced downstream signaling, particularly increased ERK1/2 phosphorylation, suggesting that PALD1 serves as a negative regulator of VEGF-A-induced signaling .

These findings collectively indicate that PALD1 functions as a phosphoinositide phosphatase that regulates endosomal trafficking of VEGFR2, thereby modulating the intensity and duration of VEGF-A-induced signaling in endothelial cells.

What methodological approaches are recommended for studying PALD1's role in angiogenesis?

Investigating PALD1's role in angiogenesis requires a multi-faceted approach combining in vitro and in vivo methodologies:

In Vivo Models:

• Postnatal retinal development model: Useful for assessing developmental angiogenesis, where PALD1-deficient mice exhibit reduced vascular outgrowth, increased filopodia extensions from endothelial tip cells, and greater vascular density at the vascular front .

• Oxygen-induced retinopathy (OIR) model: Appropriate for studying pathological angiogenesis, where PALD1-deficient mice show increased vascular tuft formation following OIR .

• Reporter mice: The Pald1+/LacZ mouse model allows tracking of Pald1 transcriptional activation in the vasculature both during development and in pathological conditions .

In Vitro Assays:

• 3D endothelial spheroid sprouting assays: Endothelial cells with PALD1 knockdown demonstrate enhanced sprouting compared to controls .

• Proliferation assays: PALD1 knockdown enhances HDMEC proliferation, supporting its role as a negative regulator of endothelial cell growth .

• Cell surface biotinylation assays: Essential for studying VEGFR2 trafficking dynamics, allowing separation and quantification of surface-localized versus internalized receptor pools following VEGF-A stimulation .

Signaling Analysis:

• MAP kinase/ERK pathway evaluation: Critical for determining downstream consequences of altered VEGFR2 trafficking. Both immunoblotting and immunostaining approaches have revealed increased ERK1/2 phosphorylation in PALD1-deficient contexts .

• Pharmacological interventions: The MEK inhibitor U0126 normalizes the vascular phenotypes observed in Pald1-deficient retinas, confirming the mechanistic importance of this pathway .

This integrated approach provides comprehensive insights into PALD1's function in both physiological and pathological angiogenesis.

What techniques can be used to effectively knockdown or knockout PALD1 for functional studies?

For effective functional analysis of PALD1, researchers have successfully employed both genetic and siRNA-based approaches:

siRNA-Mediated Knockdown:

• Human dermal microvascular endothelial cells (HDMEC) can be effectively transfected with siRNA targeting PALD1 .

• Optimal protocol involves:

  • Transfection of cells with PALD1-targeting or non-targeting control siRNAs

  • Maintenance for 72 hours at 37°C and 5% CO2

  • Serum starvation for 3 hours prior to VEGF-A stimulation

  • Validation of knockdown efficiency via Western blot or qPCR

This approach is particularly valuable for in vitro studies examining acute effects of PALD1 depletion on cellular processes.

Genetic Models:

• Pald1-/- mouse models provide a system for studying complete PALD1 deficiency in vivo .

• Pald1+/LacZ reporter mice enable visualization of PALD1 expression patterns through β-galactosidase activity .

These genetic approaches offer advantages for studying PALD1 function in complex developmental or disease contexts where long-term absence of the protein is required.

When designing knockdown/knockout experiments, researchers should consider:

• Including appropriate controls (non-targeting siRNA, wild-type littermates)

• Confirming knockdown/knockout efficiency at both mRNA and protein levels

• Potential compensatory mechanisms that may arise in constitutive knockout models

• Cell type-specific differences in transfection efficiency when using siRNA approaches

How does PALD1 deficiency affect pathological angiogenesis?

PALD1 deficiency significantly impacts pathological angiogenesis, particularly in models of retinal neovascularization. Key findings from research using the oxygen-induced retinopathy (OIR) model include:

• Mice lacking Pald1 (Pald1-/-) exhibit increased vascular tuft formation at postnatal day 17 (P17) following OIR, although the avascular area remains comparable to wild-type mice .

• PALD1 expression itself is regulated during pathological angiogenesis, with the Pald1+/LacZ reporter model demonstrating endothelial LacZ expression in both normal retinal vasculature and pathological blood vessels following OIR .

• VEGF-A specifically induces Paladin expression in endothelial cells both in vitro and in vivo, whereas other angiogenic factors such as fibroblast growth factor-2 (FGF2) or stromal derived factor 1a (SDF1a) do not .

• The mechanism underlying enhanced pathological angiogenesis in PALD1-deficient conditions appears to involve increased VEGFR2 internalization and enhanced ERK1/2 signaling .

These findings suggest that PALD1 functions as an endogenous brake on excessive angiogenesis during pathological conditions, potentially through modulation of VEGF-A/VEGFR2 signaling intensity and duration. This makes PALD1 a potential therapeutic target for conditions characterized by abnormal vascular growth, such as retinopathies, tumor angiogenesis, or inflammatory disorders with vascular components.

What strategies can be used to validate the specificity of PALD1 antibodies?

Thorough validation of PALD1 antibodies is essential for reliable research outcomes. A comprehensive validation strategy should include:

Genetic Validation:

• Testing antibodies in PALD1 knockout or knockdown models, where specific signals should be absent or significantly reduced

• Comparison of staining patterns in wild-type versus Pald1-/- tissues or cells

Multiple Antibody Approach:

• Utilizing antibodies targeting different PALD1 epitopes to confirm consistent staining patterns

• Comparing commercial antibodies from different vendors (e.g., Atlas Antibodies, Elabscience, St John's Laboratory)

Peptide Competition Assays:

• Pre-incubating the antibody with its specific immunogen (e.g., recombinant fusion protein containing amino acids 1-300 of human PALD1 for STJ114759)

• Observing elimination or substantial reduction of staining in pre-absorbed samples

Expression Pattern Analysis:

• Confirming that antibody staining matches the expected cytoplasmic localization

• Verifying tissue distribution patterns consistent with known PALD1 expression (endothelial cells, mural cells, etc.)

Western Blot Verification:

• Confirming detection of bands at the expected molecular weights (95 kDa and 140 kDa)

• Assessing signal reduction following PALD1 knockdown/knockout

Cross-Reactivity Assessment:

• Testing antibodies across multiple species if multi-species reactivity is claimed

• Confirming specificity against related proteins, particularly the PALD1 paralog PTPDC1

Implementation of these validation strategies ensures confidence in experimental results and facilitates meaningful comparisons across studies using different PALD1 antibodies.

How can researchers optimize PALD1 immunoprecipitation protocols?

Optimization of immunoprecipitation (IP) protocols for PALD1 requires careful consideration of several technical aspects:

Buffer Selection and Optimization:

• For cytoplasmic proteins like PALD1, use non-denaturing lysis buffers containing mild detergents such as NP-40 or Triton X-100 (0.1-1%)

• Include protease and phosphatase inhibitors to preserve protein integrity and phosphorylation status

• Consider buffer ionic strength (150-300 mM NaCl typical) to balance protein extraction efficiency with maintenance of protein-protein interactions

Antibody Selection:

• Choose antibodies validated for IP applications, such as those demonstrated to successfully pull down PALD1

• Consider using antibodies that recognize native protein conformations rather than denatured epitopes

• Optimal antibody amounts range from 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

Control Implementation:

• Include isotype control antibodies to assess non-specific binding

• Where available, include PALD1-deficient samples as negative controls

• Consider pre-clearing lysates with protein A/G beads to reduce background

Protocol Considerations:

• Pre-form antibody-bead complexes before adding lysate to improve capture efficiency

• Optimize incubation times (typically 1-4 hours or overnight at 4°C)

• Use gentle washing conditions to preserve specific interactions while removing background

• For co-immunoprecipitation studies aimed at identifying PALD1 interaction partners, consider cross-linking approaches to stabilize transient interactions

Detection Methods:

• For Western blot detection after IP, use different antibodies for IP and detection when possible to avoid heavy/light chain interference

• Consider using TrueBlot® or similar secondary antibodies that preferentially detect native (non-denatured) IgG to minimize heavy/light chain signal

• For mass spectrometry analysis, optimize elution conditions to maximize protein recovery

Successful implementation of these strategies facilitates reliable investigation of PALD1 protein complexes and interacting partners.

What are the emerging areas of PALD1 research in disease contexts?

Based on current evidence, several promising directions for PALD1 research in disease contexts are emerging:

• Neurodegenerative diseases: The association between PALD1 and Alzheimer Disease 7 suggests potential roles in neurodegeneration that remain largely unexplored. Future studies might investigate how PALD1 influences neuronal signaling, microglial function, or neurovascular interactions.

• Cancer biology: Given PALD1's role in angiogenesis regulation and its expression in various cancer tissues (colon cancer, breast cancer, ovary tumor) , investigations into how PALD1 affects tumor vascularization, progression, and metastasis represent important research opportunities.

• Retinal pathologies: The demonstrated role of PALD1 in pathological retinal angiogenesis suggests potential therapeutic applications in diabetic retinopathy, age-related macular degeneration, and other vascular retinopathies.

• Vascular development disorders: The involvement of PALD1 in developmental angiogenesis indicates possible roles in congenital vascular anomalies that warrant further investigation.

• Inflammatory conditions: As PALD1 is expressed in peripheral blood mononuclear cells , research into its potential immunomodulatory functions could reveal roles in inflammatory or autoimmune conditions.

Addressing these research areas will require continued development and refinement of PALD1-specific research tools, including selective inhibitors, tissue-specific conditional knockout models, and advanced imaging techniques to visualize PALD1 dynamics in living systems.

What technologies are on the horizon for studying PALD1 function?

Several cutting-edge technologies offer promising approaches for advancing PALD1 research:

• CRISPR-Cas9 genome editing: Beyond simple knockout models, precision engineering of PALD1 domains or phosphorylation sites could provide mechanistic insights into specific protein functions.

• Super-resolution microscopy: Techniques such as STORM, PALM, or STED microscopy could reveal PALD1's precise subcellular localization and dynamics during endosomal trafficking with unprecedented detail.

• Proximity labeling approaches: BioID or APEX2-based systems could identify the PALD1 interactome in specific cellular compartments and under various stimulation conditions.

• Phosphoproteomics: Comprehensive analysis of how PALD1 deficiency affects cellular phosphorylation networks could reveal broader impacts on signaling beyond the established VEGFR2-ERK axis.

• Patient-derived organoids: These systems could enable investigation of PALD1 function in human disease contexts, particularly in vascular disorders or cancer.

• In vivo imaging: Development of PALD1 activity reporters or biosensors could enable real-time visualization of its phosphatase activity in living systems.

Integration of these technologies with established models will likely accelerate understanding of PALD1's multifaceted roles in cellular physiology and disease pathogenesis.