PIK3AP1 Antibody

What is PIK3AP1 and what is its role in cellular signaling?

PIK3AP1, also known as B-cell adaptor for phosphoinositide 3-kinase (BCAP), functions as an adaptor protein that links B cell receptor signaling to the phosphoinositide 3-kinase (PI3K) pathway. PIK3AP1 plays a critical role in the PI3K-Akt signaling cascade, which is essential for various cellular processes including cell growth, proliferation, metabolism regulation, and inhibition of apoptosis . The protein is particularly important in immune cells, where it helps transduce signals from membrane receptors to intracellular effectors. Recent studies have demonstrated that PIK3AP1 is involved in autophagy regulation through its effect on the AKT/mTOR pathway .

What types of PIK3AP1 antibodies are available for research purposes?

Based on current research resources, PIK3AP1 antibodies are available in several formats with varying specifications:

These antibodies recognize different epitopes of PIK3AP1 and vary in their applications, making selection dependent on specific experimental requirements.

How does PIK3AP1 interact with the PI3K-Akt pathway?

PIK3AP1 serves as an adaptor protein that facilitates activation of the PI3K-Akt pathway following receptor stimulation. Upon activation, PIK3AP1 undergoes phosphorylation, creating binding sites for the p85 regulatory subunit of PI3K. This interaction positions PI3K in proximity to its substrate, phosphatidylinositol-4,5-bisphosphate (PIP2), which is then converted to phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 subsequently recruits and activates Akt, triggering downstream signaling events that regulate cellular processes such as metabolism, growth, proliferation, and survival .

Research has demonstrated that interference with PIK3AP1 expression through siRNA knockdown or miRNA regulation affects phosphorylation levels of AKT and mTOR, confirming its role in modulating this pathway. Specifically, experiments with A549 and LLC cells showed that when PIK3AP1 was silenced, phosphorylation of both AKT and mTOR was reduced .

How can PIK3AP1 antibodies be used to study viral infection mechanisms?

Recent research has identified PIK3AP1 as a host factor involved in viral infection mechanisms, particularly in African swine fever virus (ASFV) infections. Quantitative proteomics using isobaric tags for relative and absolute quantitation (iTRAQ) combined with liquid chromatography-mass spectrometry (LC-MS/MS) revealed that PIK3AP1 is among the differentially expressed proteins during ASFV infection of bone marrow-derived macrophages .

Researchers can utilize PIK3AP1 antibodies to:

• Monitor changes in PIK3AP1 expression levels during viral infection using Western blotting

• Investigate protein-protein interactions between viral proteins and PIK3AP1 using co-immunoprecipitation

• Visualize subcellular localization changes of PIK3AP1 during infection using immunofluorescence

Interestingly, overexpression of PIK3AP1 has been shown to inhibit ASFV replication independent of the PI3K-Akt pathway. Additionally, the viral protein MGF360-9L interacts with PIK3AP1 and decreases its protein expression level, suggesting a viral evasion mechanism that targets this host defense factor .

What experimental controls should be included when using PIK3AP1 antibodies in Western blotting experiments?

When conducting Western blotting experiments with PIK3AP1 antibodies, researchers should implement the following controls to ensure reliability and validity of results:

• Positive control: Include lysates from cells known to express PIK3AP1, such as mouse thymus tissue, HeLa cells, or Jurkat cells .

• Negative control: Either use lysates from cells with PIK3AP1 knockdown, or omit the primary antibody while maintaining all other steps.

• Loading control: Include antibodies against housekeeping proteins (e.g., GAPDH, β-actin) to normalize protein loading across lanes.

• Molecular weight marker: Confirm that the detected band appears at the expected molecular weight for PIK3AP1 (approximately 97-100 kDa).

• Antibody validation: When first using a new PIK3AP1 antibody, consider running a validation test using recombinant PIK3AP1 protein to confirm specificity.

For quantitative Western blot analysis, it is recommended to optimize antibody concentrations. For example, the polyclonal rabbit anti-PIK3AP1 antibody can be used at 0.5-3μg/mL , while mouse monoclonal antibodies may require different concentrations as specified by manufacturers.

How does miR-486 regulation of PIK3AP1 impact cellular autophagy?

Research has revealed that miR-486 directly regulates post-transcriptional expression of PIK3AP1. This microRNA-mediated regulation has significant implications for cellular autophagy—a critical process for cellular homeostasis and stress response .

Experimental evidence indicates that:

• Transfection of cells with miR-486 mimic reduces both mRNA and protein levels of PIK3AP1

• Luciferase reporter assays confirm direct binding of miR-486 to the 3'-UTR of PIK3AP1

• Silencing of PIK3AP1 (via siRNA) or overexpression of miR-486 increases autophagosome formation

• The increase in autophagy is associated with decreased phosphorylation of AKT and mTOR

Importantly, co-transfection with PIK3AP1 expression plasmid rescues the effects of miR-486 on autophagy and signaling pathway activation, confirming the specificity of the interaction. Researchers studying autophagy regulation can utilize PIK3AP1 antibodies in Western blot analysis to monitor these changes in the AKT/mTOR pathway following miR-486 manipulation .

What are the optimal storage conditions for PIK3AP1 antibodies and how can their stability be maintained?

To maintain the stability and functionality of PIK3AP1 antibodies, researchers should follow these storage recommendations:

• Short-term storage: Store at 4°C for frequent use over a period of days to weeks.

• Long-term storage: Aliquot and store at -20°C for up to 24 months to minimize freeze-thaw cycles.

• Freeze-thaw cycles: Avoid repeated freeze-thaw cycles as these can degrade antibody quality and reduce sensitivity. Create multiple small aliquots before freezing.

• Buffer considerations: PIK3AP1 antibodies are typically supplied in phosphate-buffered saline (PBS, pH 7.4) containing preservatives such as 0.05% Proclin-300 and stabilizers like 50% glycerol .

Stability assessments indicate that properly stored PIK3AP1 antibodies show less than 5% loss rate within the expiration date, as determined by accelerated thermal degradation tests (37°C for 48 hours) . Always check manufacturer-specific recommendations, as formulations may vary.

What is the recommended protocol for optimizing immunohistochemistry experiments using PIK3AP1 antibodies?

When optimizing immunohistochemistry (IHC) experiments with PIK3AP1 antibodies, researchers should follow this systematic approach:

• Antibody dilution optimization: Begin with the manufacturer's recommended range (e.g., 5-30μg/mL for polyclonal anti-PIK3AP1) and prepare a dilution series to determine optimal working concentration.

• Antigen retrieval method selection:

  • Heat-induced epitope retrieval (HIER): Test both citrate buffer (pH 6.0) and EDTA buffer (pH 9.0)

  • Enzymatic retrieval: Consider proteinase K digestion if heat-based methods prove insufficient

• Blocking optimization: Test different blocking solutions (e.g., 5% BSA, 5% normal serum from the same species as the secondary antibody) and incubation times (30-60 minutes).

• Primary antibody incubation: Compare different incubation conditions:

  • Room temperature (1-2 hours) vs. 4°C (overnight)

  • Humidity chamber to prevent drying

• Detection system selection: For PIK3AP1 detection, HRP-conjugated secondary antibodies (e.g., HRP-Linked Caprine Anti-Rabbit IgG at 2μg/mL) with DAB substrate have been successfully used .

• Counterstaining: Adjust hematoxylin counterstaining time to achieve appropriate nuclear staining without obscuring specific DAB signal.

• Controls: Always include positive control tissue (e.g., human stomach tissue has shown PIK3AP1 expression) , negative control (primary antibody omitted), and isotype control.

An optimized protocol should yield clean, specific staining with minimal background and reproducible results across experimental replicates.

How can researchers troubleshoot non-specific binding issues when using PIK3AP1 antibodies in immunofluorescence studies?

When encountering non-specific binding in immunofluorescence experiments with PIK3AP1 antibodies, researchers should implement the following troubleshooting strategies:

• Increase blocking stringency:

  • Extend blocking time from 1 hour to 2 hours

  • Increase BSA concentration from 5% to 10%

  • Add 0.1-0.3% Triton X-100 to the blocking solution to reduce hydrophobic interactions

• Optimize antibody dilution:

  • For PIK3AP1 antibodies, test dilutions within the recommended range (5-20μg/mL)

  • Prepare a series of dilutions to identify the concentration that maximizes specific signal while minimizing background

• Modify washing protocol:

  • Increase number of washes (5-6 times for 5 minutes each)

  • Add 0.05-0.1% Tween-20 to washing buffer

  • Use gentle agitation during washing steps

• Pre-adsorb the antibody:

  • Incubate the diluted primary antibody with 5% of the host animal serum for 1 hour before application

  • This helps remove antibodies that might cross-react with endogenous immunoglobulins

• Secondary antibody considerations:

  • Use highly cross-adsorbed secondary antibodies

  • Select fluorophores with spectral properties distinct from any autofluorescence in the sample

  • Validate secondary antibody specificity by omitting primary antibody

• Image acquisition parameters:

  • Optimize exposure settings to prevent oversaturation

  • Use appropriate filter sets to minimize bleed-through

Researchers have successfully used Alexa Fluor 488 anti-rabbit/mouse and Alexa Fluor 594 anti-rabbit/mouse IgG H&L for PIK3AP1 detection in confocal microscopy applications .

What role does PIK3AP1 play in viral pathogenesis and how might this inform antiviral strategies?

Recent studies have identified PIK3AP1 as a significant host factor in viral infections, particularly in the context of African swine fever virus (ASFV). Quantitative proteomic analysis revealed that PIK3AP1 expression is upregulated following ASFV infection of bone marrow-derived macrophages .

Key findings regarding PIK3AP1's role in viral pathogenesis include:

• Antiviral activity: Overexpression of PIK3AP1 significantly inhibits ASFV replication in vitro.

• Pathway independence: Surprisingly, the antiviral effect of PIK3AP1 operates independently of the PI3K-Akt pathway, suggesting an alternative mechanism of action.

• Viral counteraction: ASFV has evolved a specific countermeasure to PIK3AP1's antiviral activity. The viral protein MGF360-9L interacts directly with PIK3AP1 and reduces its protein expression level.

• Pathway manipulation: Despite PIK3AP1's PI3K-independent antiviral activity, the PI3K-Akt pathway itself promotes ASFV replication, as demonstrated by reduced viral replication when the pathway is inhibited by LY294002.

These findings suggest potential antiviral strategies:

• Developing compounds that enhance PIK3AP1 stability or expression

• Creating peptide inhibitors that disrupt the interaction between MGF360-9L and PIK3AP1

• Combining PIK3AP1-targeted approaches with PI3K-Akt pathway inhibitors for synergistic effects

This research provides a foundation for understanding host-virus interactions and developing novel antiviral therapeutics targeting PIK3AP1-related mechanisms .

How does the relationship between PIK3AP1 and autoimmune diseases inform antibody-based research approaches?

While the search results do not provide extensive information about PIK3AP1 and autoimmune diseases directly, there is an indication of related pathways being involved in systemic lupus erythematosus (SLE). The B-cell scaffold protein with ankyrin repeats 1 (BANK1), which operates in a similar cellular context as PIK3AP1, has confirmed genetic associations with SLE .

Given the role of PIK3AP1 in B-cell receptor signaling and its connection to the PI3K pathway, researchers investigating autoimmune diseases should consider:

• Comparative expression analysis: Using PIK3AP1 antibodies to evaluate expression levels in immune cells from autoimmune disease patients versus healthy controls.

• Genetic association studies: Investigating whether PIK3AP1 has genetic variants similar to the SLE-associated SNPs found in BANK1 (rs17266594 and rs10516487) .

• Functional studies: Examining how PIK3AP1 affects B-cell activation, antibody production, and cytokine release in autoimmune disease models.

• Pathway crosstalk: Investigating potential interactions between PIK3AP1 and other known autoimmunity-associated proteins like BANK1.

• Therapeutic targeting: Exploring whether modulation of PIK3AP1 expression or function could affect autoimmune disease progression.

These approaches would benefit from well-validated PIK3AP1 antibodies suitable for various applications including Western blotting, flow cytometry, immunohistochemistry, and co-immunoprecipitation studies.

What insights have proteomics studies provided regarding PIK3AP1 regulation and function?

Proteomic approaches have yielded significant insights into PIK3AP1 regulation and function in various cellular contexts. Using isobaric tags for relative and absolute quantitation (iTRAQ) combined with liquid chromatography-mass spectrometry (LC-MS/MS), researchers have identified PIK3AP1 as one of the differentially expressed proteins during viral infection .

Key proteomic findings include:

• Expression dynamics: PIK3AP1 is among 286 upregulated proteins identified in bone marrow-derived macrophages following ASFV infection, suggesting its involvement in host response mechanisms.

• Post-transcriptional regulation: Proteomic studies combined with transcriptomic analyses have revealed that PIK3AP1 is subject to microRNA-mediated regulation. Specifically, miR-486 directly targets the 3'-UTR of PIK3AP1, affecting its protein expression levels .

• Protein-protein interactions: Proteomics approaches have identified interaction partners of PIK3AP1, including the viral protein MGF360-9L, which physically interacts with PIK3AP1 and decreases its expression .

• Pathway mapping: Quantitative proteomics has helped position PIK3AP1 within signaling networks, confirming its role in PI3K-Akt pathway regulation while also suggesting PI3K-independent functions.

These proteomic insights provide researchers with valuable direction for antibody-based studies, indicating which cell types, conditions, and potential interaction partners should be prioritized when investigating PIK3AP1 function.

What experimental approaches are most effective for studying PIK3AP1 protein-protein interactions?

For researchers investigating PIK3AP1 protein-protein interactions, several complementary experimental approaches have proven effective:

• Co-immunoprecipitation (Co-IP):

  • Use anti-PIK3AP1 antibodies (3-5μg per reaction) to pull down PIK3AP1 and associated proteins

  • Analyze precipitated complexes by Western blotting or mass spectrometry

  • This approach successfully identified the interaction between PIK3AP1 and viral protein MGF360-9L

• Proximity labeling methods:

  • BioID or TurboID fusion proteins can identify transient or weak interactors

  • APEX2-based proximity labeling for spatially resolved interaction mapping

  • These methods complement traditional Co-IP by capturing interactions in their native cellular context

• Fluorescence resonance energy transfer (FRET):

  • Tag PIK3AP1 and potential interactors with appropriate fluorophore pairs

  • Analyze interaction by microscopy or flow cytometry

  • Particularly useful for studying dynamic interactions in living cells

• Split reporter protein complementation:

  • Systems like split-GFP or NanoBiT can validate direct protein-protein interactions

  • Provides spatial information about where in the cell the interaction occurs

• Yeast two-hybrid screening:

  • Useful for identifying novel interaction partners

  • Requires validation in mammalian systems due to potential false positives

When designing these experiments, researchers should consider:

• Using both N- and C-terminal tags to minimize interference with protein interactions

• Including appropriate controls (non-interacting proteins, interaction-deficient mutants)

• Validating key interactions with multiple complementary methods

• Considering the dynamic nature of interactions (stimulus-dependent, cell cycle-dependent)

The choice between these methods depends on the specific research question, available resources, and the nature of the interactions being studied.

How should researchers normalize and quantify PIK3AP1 expression data from Western blot experiments?

For accurate normalization and quantification of PIK3AP1 expression data from Western blot experiments, researchers should follow these methodological guidelines:

• Loading control selection:

  • Use housekeeping proteins like GAPDH, β-actin, or α-tubulin as primary normalization controls

  • Consider the experimental context—for example, GAPDH has been successfully used as a loading control in PIK3AP1 studies examining AKT/mTOR pathway activation

  • Validate that treatment conditions do not affect loading control expression

• Signal detection optimization:

  • Use appropriate antibody concentrations (0.5-3μg/mL for polyclonal anti-PIK3AP1)

  • Ensure signal is within the linear range of detection by testing multiple exposure times

  • Avoid signal saturation which prevents accurate quantification

• Quantification methodology:

  • Use dedicated image analysis software (ImageJ, Image Lab, etc.)

  • Define regions of interest consistently across all bands

  • Subtract background using local background correction methods

  • Calculate relative expression using the formula:

    • Relative PIK3AP1 expression = (PIK3AP1 signal / Loading control signal) / (Control PIK3AP1 signal / Control loading control signal)

• Technical considerations:

  • Run biological replicates (minimum n=3) on separate gels with identical conditions

  • Include an internal calibration sample on each gel to allow inter-gel normalization

  • Use the same exposure settings when comparing across experimental conditions

• Statistical analysis:

  • Apply appropriate statistical tests (t-test for two conditions, ANOVA for multiple conditions)

  • Report both mean values and measures of variation (standard deviation or standard error)

  • Consider using non-parametric tests if normality assumptions are violated

By following these guidelines, researchers can generate reliable quantitative data on PIK3AP1 expression changes in response to experimental manipulations.

What are the key considerations when designing experiments to investigate PIK3AP1's role in the PI3K-Akt pathway?

When designing experiments to investigate PIK3AP1's role in the PI3K-Akt pathway, researchers should address these critical considerations:

• Cell type selection:

  • Choose cell types with functional PI3K-Akt signaling (e.g., A549, LLC cells)

  • Consider immune cells like B cells where PIK3AP1/BCAP has established physiological relevance

  • Include both cell lines and primary cells to confirm physiological relevance

• Manipulation strategies:

  • Loss-of-function: siRNA knockdown (siBCAP) has been successfully used to reduce PIK3AP1 expression

  • Gain-of-function: Overexpression using PIK3AP1 expression plasmids

  • Domain-specific mutations: Create constructs with mutations in specific functional domains to dissect mechanism

• Pathway stimulation and inhibition:

  • Use appropriate stimuli (growth factors, receptor agonists) to activate the pathway

  • Include PI3K inhibitors (e.g., LY294002) as controls and for epistasis experiments

  • Test pathway-specific vs. PIK3AP1-specific effects by comparing PIK3AP1 manipulation with direct pathway inhibition

• Readout selection:

  • Proximal readouts: Phosphorylation of PI3K subunits

  • Intermediate readouts: Phosphorylation of AKT (Ser473, Thr308)

  • Distal readouts: Phosphorylation of mTOR and downstream targets (S6K, 4E-BP1)

  • Functional outcomes: Autophagy (GFP-LC3 puncta quantification) , cell proliferation, survival

• Temporal considerations:

  • Include multiple time points to capture both rapid (minutes) and delayed (hours) responses

  • Consider pulse-chase experiments to assess pathway dynamics

  • Monitor signaling kinetics by time-course Western blotting

• Context-dependent effects:

  • Test under various cellular conditions (normal growth, serum starvation, stress)

  • Investigate interplay with other pathways through combinatorial stimulation/inhibition

  • Examine effects in disease-relevant contexts (e.g., viral infection, where PIK3AP1 shows PI3K-independent functions)

By addressing these considerations, researchers can comprehensively characterize PIK3AP1's functions within and beyond the canonical PI3K-Akt pathway, potentially revealing novel therapeutic targets.

What are the most promising future directions for PIK3AP1 antibody-based research?

Based on current findings and technological developments, several promising directions for PIK3AP1 antibody-based research emerge:

• Single-cell analysis: Utilizing PIK3AP1 antibodies in single-cell proteomics and CyTOF (mass cytometry) to understand cell-type specific expression patterns and heterogeneity in immune cell populations and disease states.

• Spatial proteomics: Combining PIK3AP1 antibodies with multiplexed immunofluorescence or imaging mass cytometry to map PIK3AP1 spatial distribution and co-localization with interaction partners at subcellular resolution.

• Therapeutic applications: Developing function-modulating antibodies that could enhance PIK3AP1's antiviral properties for therapeutic applications, particularly given its identified role in restricting ASFV replication .

• Post-translational modifications: Generating modification-specific antibodies (phospho-PIK3AP1, ubiquitinated-PIK3AP1) to study regulatory mechanisms controlling PIK3AP1 function.

• Cross-disease comparisons: Using validated PIK3AP1 antibodies to examine expression patterns across different disease states, including viral infections, autoimmune diseases, and cancer.

• Structure-function studies: Combining antibody epitope mapping with structural biology approaches to better understand PIK3AP1 domain functions and develop more specific research tools.

• Pathway-independent functions: Further investigating the PI3K-independent functions of PIK3AP1, particularly in viral defense mechanisms, which could reveal novel signaling paradigms .

These directions represent significant opportunities for advancing our understanding of PIK3AP1 biology and translating that knowledge into potential therapeutic applications.

How can researchers integrate PIK3AP1 antibody-based assays with other omics approaches?

Integrating PIK3AP1 antibody-based assays with other omics techniques creates powerful multi-dimensional analyses that can provide comprehensive insights into PIK3AP1 function and regulation:

• Antibody-based proteomics + Transcriptomics:

  • Correlate PIK3AP1 protein expression (Western blot/IHC) with mRNA levels (RNA-seq)

  • Identify post-transcriptional regulation mechanisms (as demonstrated with miR-486)

  • Design targeted validation experiments for differentially expressed genes in PIK3AP1-modulated systems

• Immunoprecipitation + Mass Spectrometry:

  • Use PIK3AP1 antibodies for immunoprecipitation followed by mass spectrometry

  • Identify and quantify protein interaction networks and complexes

  • Discover novel interaction partners beyond known PI3K pathway components

• ChIP-seq + Antibody-based functional assays:

  • Investigate how transcription factors regulated by PIK3AP1-dependent pathways affect gene expression

  • Connect PIK3AP1 signaling to epigenetic modifications through integrated analysis

  • Map signaling pathway outputs to transcriptional responses

• Phosphoproteomics + PIK3AP1 functional studies:

  • Combine global phosphoproteomic analysis with PIK3AP1 manipulation (overexpression/knockdown)

  • Map downstream phosphorylation cascades dependent on PIK3AP1 activity

  • Identify novel substrates in the PI3K-Akt pathway and beyond

• Spatial transcriptomics + Immunofluorescence:

  • Correlate PIK3AP1 protein localization with spatially resolved transcriptomes

  • Understand tissue microenvironment effects on PIK3AP1 function

• Integration with clinical data:

  • Correlate PIK3AP1 expression patterns with patient outcomes and treatment responses

  • Stratify patient populations based on PIK3AP1-related pathway activation