Phospho-PRKD1 (S910) Antibody

What is Phospho-PRKD1 (S910) Antibody and what does it detect?

Phospho-PRKD1 (S910) antibody is a polyclonal antibody raised against a synthetic phosphopeptide derived from human PRKD1 around the phosphorylation site of Serine 910. This antibody specifically detects PRKD1 protein only when it is phosphorylated at the S910 residue, which serves as a critical marker of PRKD1 activation in cell signaling pathways . The antibody is typically generated in rabbits and demonstrates reactivity to human, mouse, and rat PRKD1 phosphorylated at this specific site. This high specificity makes it an invaluable tool for monitoring PRKD1 activation status in various experimental systems without cross-reactivity with other proteins .

The detection of phosphorylated S910 is particularly significant because this autophosphorylation event closely correlates with PRKD1 kinase activity. When investigating signaling cascades involving PRKD1, researchers can use this antibody to determine whether and when PRKD1 becomes activated in response to various stimuli or experimental interventions.

What are the recommended dilutions and applications for Phospho-PRKD1 (S910) Antibody?

The optimal dilution of Phospho-PRKD1 (S910) antibody varies by application and should be determined empirically for each experimental system. Based on manufacturer recommendations and published studies, the following dilutions serve as starting points:

For Western blotting applications, which represent the most validated use of this antibody, researchers typically employ the following protocol elements:

• Primary antibody incubation: Overnight at 4°C

• Secondary antibody: Anti-rabbit IgG (typically horseradish peroxidase-conjugated)

• Secondary antibody dilution: 1:2000-1:5000 (goat anti-rabbit)

• Detection method: Enhanced chemiluminescence systems

The working dilution should be optimized based on signal intensity, background levels, and specific experimental conditions . When transitioning between different lots or suppliers of the antibody, revalidation of optimal dilutions is strongly recommended.

How should Phospho-PRKD1 (S910) Antibody be stored and handled for optimal results?

Proper storage and handling are critical for maintaining antibody performance and reproducibility in experiments. Based on manufacturer recommendations:

Long-term storage:

• Store at -20°C for up to one year

• Aliquot upon receipt to minimize freeze-thaw cycles

• Avoid repeated freezing and thawing which significantly degrades antibody quality

Short-term storage:

• For frequent use, store aliquots at 4°C for up to one month

Buffer conditions:

• Typically supplied in PBS (pH 7.2-7.4) with:

  • 50% glycerol (cryoprotectant)

  • 0.02% sodium azide (preservative)

  • Sometimes contains 0.5% BSA (stabilizer)

Critical handling considerations:

• Centrifuge briefly before opening vials to collect liquid at the bottom

• Use sterile techniques when handling to prevent contamination

• Always use properly calibrated pipettes for accurate dilutions

• Return to recommended storage temperature promptly after use

• Record date of first use and track number of freeze-thaw cycles

Following these guidelines will help maintain antibody performance and extend its usable lifetime, ensuring consistent experimental results and reducing variability between assays.

How can researchers validate the specificity of Phospho-PRKD1 (S910) Antibody in their experimental systems?

Rigorous validation of phospho-specific antibodies is essential for generating reliable data. To validate Phospho-PRKD1 (S910) antibody specificity in your experimental system, employ the following comprehensive approach:

1. Stimulation Controls:
Treat cells with known PRKD1 activators and compare to basal conditions:

• PMA (phorbol ester): 100nM for 15-30 minutes

• Thrombin: 5U for rapid activation in endothelial cells

• Hydrogen peroxide: Strong inducer of PKD1 activation

• Polytrauma cocktail (PTC): Combination of IL1β, IL6, CXCL8, C3a, and C5a-des-Arg for endothelial cells

2. Phosphatase Treatment Control:

• Divide lysates and treat one portion with lambda phosphatase

• Western blot both samples - phosphatase treatment should eliminate the signal

3. Genetic Modulation:

• Knockdown PRKD1 using siRNA or shRNA approaches

• Example from literature: PRKD1-targeting siRNA (#5587) effectively reduces PRKD1 expression

• Signal intensity should decrease proportionally to knockdown efficiency

4. Phospho-deficient Mutant:

• Express wild-type PRKD1 alongside a S910A mutant

• Stimulate cells and immunoblot - the antibody should detect only wild-type protein

5. Peptide Competition:

• Pre-incubate antibody with the immunizing phosphopeptide

• This should abolish specific binding in Western blot or immunostaining

6. Cross-validation with Multiple Antibodies:

• Use antibodies from different suppliers targeting the same phosphorylation site

• Consistent results across antibodies strengthen confidence in specificity

7. Orthogonal Detection Methods:

• Confirm phosphorylation at S910 using mass spectrometry

• This provides sequence-level validation of the specific modification

A comprehensive example from the literature demonstrates this validation approach: researchers studying PRKD1-mediated phosphorylation of SSH1L confirmed specificity by showing that S978A mutation abrogated detection by a phospho-specific antibody, verifying both antibody specificity and the phosphorylation site .

What experimental considerations are critical when studying PRKD1 activation kinetics using Phospho-PRKD1 (S910) Antibody?

When designing experiments to study PRKD1 activation kinetics using Phospho-PRKD1 (S910) antibody, researchers must consider several critical factors that influence experimental outcomes:

1. Time Course Design:
PRKD1 activation kinetics are highly stimulus and cell-type dependent. For example, BPA-induced PRKD1 phosphorylation shows distinctly different patterns across cell types:

• MCF-7 cells: Maximal effect at 1 hour, stable for 2-4 hours

• Hs578t cells: Maximal effect at 20-30 minutes, returns to baseline by 1 hour

Recommended time points for comprehensive kinetic analysis:

• Early: 5, 15, 30 minutes

• Intermediate: 1, 2 hours

• Late: 4, 8, 24 hours

2. Dose-Response Relationships:
Establish full dose-response curves as effects can be non-linear. For example:

• BPA stimulates PRKD1 phosphorylation dose-dependently (10^-12 to 10^-7 M)

• Maximal effects observed at 10^-9 to 10^-11 M

• Higher concentrations may show diminished effects due to receptor desensitization

3. Normalization Strategy:
For accurate quantification, implement proper controls:

• Normalize phospho-S910 signal to total PRKD1 expression

• Include housekeeping controls (β-actin, GAPDH)

• Quantify using integrated density measurements

4. Cell Type Considerations:
PRKD1 activation mechanisms vary significantly between cell types:

• Epithelial vs. endothelial cells show different activation patterns

• Primary cells vs. cell lines may have different signaling kinetics

• Consider tissue-specific regulatory mechanisms

5. Technical Replication:

• Minimum of three independent biological replicates

• Technical replicates within each experiment

• Statistical analysis using appropriate tests (e.g., ANOVA with post-hoc tests)

6. Complementary Phosphorylation Sites:
PRKD1 activation involves multiple phosphorylation events:

• Monitor both S738/742 and S910 phosphorylation in parallel

• Different phosphorylation sites may show distinct kinetics

• Complete activation assessment requires monitoring multiple sites

A representative experimental data set for PRKD1 activation kinetics is shown in Figure 1 from research by Fechtner et al., which demonstrates time-dependent phosphorylation of PRKD1 at S910 in response to inflammatory stimuli in endothelial cells .

How does PRKD1 S910 phosphorylation contribute to endothelial inflammation and how can researchers investigate this mechanism?

PRKD1 has emerged as a critical regulator of endothelial inflammation, with S910 phosphorylation serving as a key indicator of its activation in inflammatory contexts. The following methodological approach enables comprehensive investigation of PRKD1's role in endothelial inflammation:

1. Detection of PRKD1 Activation in Inflammatory Contexts:
PRKD1 becomes rapidly phosphorylated at S910 in endothelial cells exposed to inflammatory stimuli, as demonstrated in multiple experimental systems:

• In vitro: HUVEC cells treated with polytrauma cocktail (PTC) or thrombin (5U) show rapid PRKD1 phosphorylation

• In vivo: Mouse lung endothelial cells exhibit increased pPRKD S916 (mouse equivalent of human S910) after thorax trauma or polytrauma with hemorrhagic shock

2. Experimental Approach to Study PRKD1-Mediated Inflammatory Signaling:

A. Pharmacological Inhibition Studies:

• Use specific PRKD inhibitors: CRT0066101 or Kb-NB-142-70 (5 μM)

• Measure effects on:

  • Adhesion molecule expression (ICAM-1, VCAM-1, E-selectin)

  • Pro-inflammatory cytokine production (IL-6, CXCL8)

  • NFκB activation (pP65 S536 phosphorylation)

B. Genetic Manipulation:

• Stable knockdown using shRNA against PRKD1

• Rescue experiments with re-expression of wild-type PRKD1

• Expression of phospho-mimetic or phospho-deficient PRKD1 mutants

C. Functional Assays:

• Neutrophil adhesion assays using fluorescently labeled PMNs

• Transendothelial migration assays to assess barrier function

• Respiratory burst assays to measure neutrophil activation

3. Key Molecular Mechanisms and Experimental Readouts:

NFκB-Dependent Gene Expression:

• PRKD1 inhibition significantly reduces expression of:

  • Adhesion molecules: ICAM-1 (70% reduction), VCAM-1 (65% reduction), E-selectin

  • Pro-inflammatory cytokines: IL-6 (80% reduction), CXCL8 (75% reduction)

4. Recommended Experimental Protocol:

For investigating PRKD1's role in endothelial-neutrophil interactions:

• Treat endothelial cells with inflammatory stimuli (PTC) ± PRKD inhibitors

• Harvest supernatants after 4-8 hours

• Add supernatants to isolated neutrophils

• Stimulate with fMLP (100 nM) for 15 minutes

• Measure neutrophil activation (respiratory burst) or adhesion

This approach has revealed that PRKD1 inhibition significantly reduces neutrophil activation and adhesion to endothelial cells, demonstrating its crucial role in coordinating endothelial-neutrophil communication during inflammation. Researchers can apply this methodology to investigate PRKD1's role in various inflammatory conditions and potential therapeutic interventions.

How can researchers differentiate between PRKD1 S910 phosphorylation and phosphorylation of related PRKD family members?

Distinguishing between phosphorylation of PRKD1 at S910 and analogous sites in other PRKD family members requires careful experimental design, as PRKD1, PRKD2, and PRKD3 share significant sequence homology and similar regulatory mechanisms. The following methodological approach ensures accurate identification of specific PRKD isoforms:

1. Antibody Selection and Validation:

Although commercial antibodies claim specificity for PRKD1 (phospho S910), cross-reactivity with other PRKD isoforms is possible. Notably, PRKD3 (phospho S910) antibodies are also commercially available , indicating conservation of this phosphorylation site across family members.

Validation hierarchy for antibody specificity:

• Confirm epitope sequence differences between PRKD isoforms

• Test against recombinant phosphorylated proteins of all PRKD isoforms

• Use isoform-specific knockdown/knockout controls

2. Molecular Weight Discrimination:

PRKD family members have distinct molecular weights that can be resolved by SDS-PAGE:

• PRKD1: ~115-120 kDa

• PRKD2: ~105 kDa

• PRKD3: ~100 kDa

Optimization for molecular weight discrimination:

• Use 7.5-8% acrylamide gels for better separation

• Include molecular weight markers with 10 kDa resolution

• Extend electrophoresis time to maximize band separation

3. Isoform-Specific Genetic Manipulation:

Employ targeted knockdown approaches to confirm antibody specificity:

• siRNA/shRNA targeting specific PRKD isoforms

  • PRKD1-targeting siRNA (#5587) has been validated in multiple studies

• CRISPR/Cas9-mediated knockout of specific PRKD genes

• Rescue experiments with isoform-specific expression constructs

Example experimental design:

• Transfect cells with siRNA targeting PRKD1, PRKD2, or PRKD3

• Stimulate cells with activators (e.g., PMA, thrombin)

• Analyze lysates by western blot with phospho-specific antibodies

• A reduction in signal only with PRKD1 knockdown confirms specificity

4. Sequential Immunoprecipitation Approach:

For definitive isoform-specific phosphorylation analysis:

• Immunoprecipitate with isoform-specific antibody (non-phospho)

• Immunoblot with phospho-specific antibody

• This confirms the phosphorylation occurs on the specific isoform

5. Cell Type Selection:

Some cell types predominantly express one PRKD isoform:

• Identify expression patterns of PRKD family members in your cell type

• Select models with predominant expression of specific isoforms

• Consider isoform-specific functions in physiological contexts

By implementing these approaches, researchers can ensure that the phosphorylation signal detected truly represents PRKD1 S910 phosphorylation and not cross-reactivity with other PRKD family members, thus avoiding misattribution of functional effects to specific isoforms.

What are the optimal stimuli for inducing PRKD1 S910 phosphorylation in different cell types?

The selection of appropriate stimuli for PRKD1 activation is critical for experimental design and varies significantly by cell type. Based on published research, here is a comprehensive guide to optimal stimuli for inducing PRKD1 S910 phosphorylation across different cellular contexts:

1. Epithelial/Cancer Cell Lines:

MCF-7 (Breast cancer cells):

• BPA (Bisphenol A): 10^-11 to 10^-8 M

  • Optimal concentration: 10^-9 to 10^-11 M

  • Peak activation: 1 hour post-treatment

  • Duration: Stable for 2-4 hours

Hs578t (Breast cancer cells):

• BPA: Similar dose-response as MCF-7

  • Peak activation: More rapid (20-30 minutes)

  • Duration: More transient (returns to baseline after 1 hour)

HEK293T cells:

• PMA: 100 nM

  • Rapid induction of phosphorylation

  • Used in validation studies for phospho-specific antibodies

A-431 (Epidermoid carcinoma cells):

• Used for antibody validation in Western blot analyses

• Demonstrates detectable basal or inducible PRKD1 phosphorylation

2. Endothelial Cells:

HUVEC (Human Umbilical Vein Endothelial Cells):

• Polytrauma cocktail (PTC): Combination of:

  • IL1β

  • IL6

  • CXCL8

  • C3a

  • C5a-des-Arg

• Thrombin: 5U

  • Rapid activation kinetics

  • Used in models of vascular injury

3. General Activators Effective Across Multiple Cell Types:

• Hydrogen peroxide (H₂O₂): Strong inducer of PKD1 activation

  • Used in biochemical validation studies

• RhoA activation: Linked to PKD1 phosphorylation

  • Important in cytoskeletal regulation pathways

• Phorbol esters (e.g., PMA, PDBu): 50-100 nM

  • Classic activators of PKC/PKD pathways

  • Widely used positive controls

4. In Vivo Activation Models:

• Thorax trauma (TxT) in mouse models

  • Induces PRKD1 phosphorylation in lung tissue

  • Detected after 4 hours post-injury

• Polytrauma with hemorrhagic shock (PT + HS)

  • Strong activation in lung microvascular endothelial cells

  • Associated with neutrophil infiltration

Experimental Design Recommendations:

When designing experiments, always include time course and dose-response analyses to determine optimal conditions for your specific experimental system, as activation kinetics can vary significantly between cell types and experimental conditions.

How can Phospho-PRKD1 (S910) Antibody be used for immunohistochemical analysis of tissue sections?

Successful immunohistochemical detection of phosphorylated PRKD1 in tissue sections requires careful optimization of multiple technical parameters. The following protocol outlines critical considerations and methodological approaches for obtaining specific, reproducible detection of PRKD1 S910 phosphorylation in tissue contexts:

1. Tissue Preparation and Fixation:

Phospho-epitopes are particularly sensitive to fixation conditions:

• Optimal fixation: 10% neutral buffered formalin for 12-24 hours

• Include phosphatase inhibitors in fixation solution

• Process tissues promptly after collection to preserve phosphorylation status

• For frozen sections, snap-freeze immediately and store at -80°C

2. Antigen Retrieval Optimization:

Phospho-epitopes often require rigorous antigen retrieval:

• Heat-induced epitope retrieval (HIER) methods:

  • Citrate buffer (pH 6.0): Start with this standard approach

  • EDTA buffer (pH 9.0): May improve retrieval of some phospho-epitopes

  • Optimization: Test multiple conditions with positive control tissues

• Pressure cooker or microwave-based retrieval (20 minutes) often yields best results

3. Blocking and Antibody Parameters:

• Blocking solution: 5-10% normal serum (from secondary antibody species)

• Add 0.1-0.3% Triton X-100 for improved antibody penetration

• Primary antibody dilution: Start with 1:500, then optimize (1:200-1:1000 range)

• Incubation conditions: Overnight at 4°C in humidified chamber

• Signal amplification: Consider tyramide signal amplification for low-abundance phospho-proteins

4. Multi-marker Analysis Strategy:

As demonstrated in published research, co-staining with cell-type specific markers provides crucial context:

• Endothelial activation: Co-stain with CD34 (endothelial marker)

• Inflammatory context: Co-stain with MPO (neutrophil marker)

• Quantification approach: Generate detection masks using cell-type markers to measure phospho-PRKD1 specifically within cell populations of interest

5. Controls for Phospho-specific IHC:

• Positive control: Tissues with known PRKD1 activation (e.g., lung tissues after trauma)

• Negative controls:

  • Phosphatase-treated sections

  • Primary antibody omission

  • Non-specific IgG substitution

  • Tissues from PRKD1 knockout models (if available)

6. Imaging and Quantification:

Appropriate imaging parameters are essential for reproducible results:

• Confocal microscopy with airy confocal sections for precise co-localization

• Equal acquisition settings across all samples and controls

• Quantitative analysis methods:

  • ROI analysis for specific cellular structures

  • Fluorescence intensity measurement within cell-type specific masks

  • Example from literature: "Quantitative mean of ROI analysis for endothelial cells was performed by generating detection masks using the CD34 signal in NIH ImageJ"

7. Species Considerations:

Important note on species differences:

• The equivalent phosphorylation site in mouse PRKD1 is S916 (human S910)

• Ensure the antibody has been validated for your species of interest

• When comparing across species literature, be aware of this numbering difference

This detailed protocol, based on published methodologies that successfully detected phospho-PRKD1 in lung tissues, provides a framework that can be adapted to various research questions involving PRKD1 activation in different tissues and disease models.