PSTPIP2 Antibody

What are the optimal applications for detecting PSTPIP2 in experimental settings?

PSTPIP2 can be detected through several established methods, with Western blotting, immunoprecipitation, and immunofluorescence being the most reliable. Based on validation data, PSTPIP2 antibodies such as 13450-1-AP have demonstrated consistent reactivity in multiple applications . For Western blotting, a dilution range of 1:500-1:1000 typically yields optimal results, while immunoprecipitation requires approximately 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate . Importantly, PSTPIP2 antibodies have been validated with human, mouse, and rat samples, though researchers should conduct application-specific optimizations as signal strength can vary between tissue types .

What is the expected molecular weight and cellular localization pattern for PSTPIP2 in immunostaining experiments?

PSTPIP2 has a calculated molecular weight of 36 kDa (312 amino acids) and typically appears as a distinct 36 kDa band in Western blot analyses . In immunofluorescence experiments, PSTPIP2 exhibits predominantly cell membrane localization, consistent with its function as an F-BAR domain-containing adaptor protein . When co-staining with cell-specific markers, researchers should expect PSTPIP2 to show particularly strong expression in myeloid lineage cells, including monocytes, macrophages, and neutrophils . In kidney tissue, PSTPIP2 is primarily expressed in distal tubule epithelial cells and collecting ducts .

How should researchers validate PSTPIP2 antibody specificity before experimental use?

A multilayered validation approach is recommended:

• Western Blot Verification: Confirm a single band at approximately 36 kDa using positive control samples such as RAW 264.7 cells

• Overexpression Controls: Include PSTPIP2-overexpressing samples (e.g., using GV230-PSTPIP2 plasmid) alongside negative controls

• Knockdown Validation: Implement siRNA or shRNA against PSTPIP2 as a negative control

• Immunoprecipitation Confirmation: Perform IP followed by mass spectrometry to verify pulled-down proteins

• Cross-Reactivity Testing: Test against similar F-BAR domain proteins to ensure specificity

This comprehensive validation strategy ensures reliable and reproducible results in downstream applications.

How can researchers effectively study PSTPIP2 expression patterns in inflammatory disease models?

To comprehensively characterize PSTPIP2 expression in inflammatory disease models, researchers should implement a multi-modal approach:

• Temporal Expression Analysis: In animal models such as collagen-induced arthritis (CIA), analyze PSTPIP2 expression at multiple timepoints (e.g., day 35 peak inflammation and day 70 remission phase) to capture dynamic changes

• Cell-Specific Quantification: Use flow cytometry with cell-specific markers (CD11b for monocytes/macrophages) combined with PSTPIP2 antibodies to quantify expression levels across different cell populations

• Tissue Distribution Assessment: Employ immunohistochemistry with tissue-specific markers, such as:

  • Kidney: LTL (proximal tubule), calbindin D28k (distal tubule), aquaporin-3 (collecting duct)

  • Synovium: CD11b (macrophages), F4/80 (tissue macrophages)

• Reporter Systems: Consider using reporter mice like Pstpip2^CreR26-ZsGreen for in vivo tracking of PSTPIP2-expressing cells

• Correlation Analysis: Correlate PSTPIP2 expression with disease severity metrics (e.g., DAS28 scores in RA patients)

This approach provides comprehensive data on PSTPIP2 expression dynamics across different disease stages and cell types.

What experimental controls are essential when studying PSTPIP2 protein interactions through co-immunoprecipitation?

When investigating PSTPIP2 protein interactions, the following controls are crucial:

• Input Control: Include 5-10% of pre-IP lysate to verify protein presence before IP

• Isotype Control: Use matched isotype antibody (rabbit IgG for 13450-1-AP) to identify non-specific binding

• Reciprocal IP: Confirm interactions by performing reverse IP with antibodies against suspected binding partners (e.g., NF-κB p65)

• Mutation Controls: Include samples expressing PSTPIP2 with mutations in key interaction domains:

  • W232A mutation to disrupt PEST-phosphatase binding

  • Y323A/Y329A/Y333A mutations to prevent SHIP1 binding

• Competitive Binding Assay: Include excess of known binding peptides to demonstrate specificity

• Crosslinking Validation: For transient interactions, use reversible crosslinkers prior to IP

These controls ensure specificity and validity of detected protein-protein interactions.

How should researchers design experiments to investigate the role of PSTPIP2 in macrophage polarization and dynamics?

To effectively study PSTPIP2's role in macrophage polarization and dynamics:

• Isolation Protocol: Implement gentle isolation of synovial macrophages using enzymatic digestion without mechanical disruption to preserve cell surface markers

• Polarization Assessment:

  • Flow cytometry panel: CD11b+CD206+ for M2 and CD11b+CD86+ for M1 macrophages

  • qRT-PCR for polarization markers: TNF-α, IL-1β (M1); IL-10, Arg1 (M2)

  • ELISA for secreted cytokines

• Dynamic Visualization:

  • Time-lapse video microscopy with Actin and PSTPIP2 co-staining

  • Quantify filopodia formation and membrane ruffling using ImageJ analysis

  • Track macrophage migration in response to inflammatory stimuli

• Genetic Manipulation:

  • Use Esr2^fl/fl/Adgre-Cre mice to study ERβ dependency

  • Employ adeno-associated virus (AAV) for PSTPIP2 overexpression in vivo

  • Implement CRISPR/Cas9 for conditional Pstpip2 knock-in

• Barrier Formation Assessment:

  • Quantify expression of tight junction proteins (Claudin5, ZO-1) in PSTPIP2+ macrophages

  • Evaluate barrier integrity through permeability assays

This comprehensive approach enables detailed characterization of PSTPIP2's role in macrophage biology.

What are the common challenges in detecting PSTPIP2 in tissue samples, and how can they be overcome?

Common challenges and solutions include:

For particularly challenging samples, signal amplification using tyramide signal amplification (TSA) can increase detection sensitivity without increasing background .

How can researchers resolve conflicting data when PSTPIP2 expression differs between protein and mRNA levels?

When faced with discrepancies between PSTPIP2 protein and mRNA expression:

• Assess Post-Transcriptional Regulation:

  • Measure microRNA levels, particularly those predicted to target PSTPIP2

  • Investigate RNA-binding proteins that may affect PSTPIP2 mRNA stability

• Evaluate Protein Stability:

  • Perform cycloheximide chase assays to determine PSTPIP2 protein half-life

  • Test proteasome inhibitors (MG132) to assess degradation pathways

• Examine Experimental Limitations:

  • Compare antibody epitopes with mRNA primer binding sites to ensure targeting the same isoforms

  • Verify the specificity of both antibodies and PCR primers using appropriate controls

• Consider Biological Context:

  • In inflammatory conditions, post-translational modifications may affect antibody recognition without altering mRNA levels

  • Examine cell-specific expression patterns as bulk analysis may mask cell-type-specific variations

• Technical Validation:

  • Employ absolute quantification methods (digital PCR for mRNA, quantitative Western blot for protein)

  • Use multiple antibodies targeting different epitopes to confirm protein expression patterns

This systematic approach can reconcile apparent contradictions between protein and mRNA expression data.

How can researchers effectively use PSTPIP2 antibodies to study its role in inflammatory disease progression?

To leverage PSTPIP2 antibodies for studying inflammatory disease progression:

• Sequential Tissue Analysis:

  • Implement serial biopsies across disease progression in animal models

  • Perform multiplexed IHC/IF with PSTPIP2 and inflammatory markers (IL-1β, NF-κB p65)

• Functional Assessment:

  • Combine PSTPIP2 detection with phospho-specific antibodies (p-NF-κB, p-IκBα) to evaluate pathway activation

  • Correlate PSTPIP2 levels with inflammatory cytokine production using in situ hybridization or multiplexed protein detection

• Therapeutic Response Monitoring:

  • Track changes in PSTPIP2 expression and localization following treatment interventions

  • Develop tissue-based assays to predict therapeutic response based on PSTPIP2 expression patterns

• Disease-Specific Applications:

  • For kidney diseases: Focus on distal tubule and collecting duct PSTPIP2 expression using segment-specific markers

  • For arthritis: Evaluate synovial lining PSTPIP2+ macrophage barrier formation

  • For osteomyelitis: Assess neutrophil PSTPIP2 expression in relation to IL-1β production

• Translational Biomarkers:

  • Establish PSTPIP2 expression thresholds that correlate with disease severity across multiple inflammatory conditions

  • Develop standardized PSTPIP2 immunoassays for patient stratification in clinical trials

These approaches facilitate comprehensive characterization of PSTPIP2's role across disease stages.

What methodological considerations are important when studying PSTPIP2 interactions with NF-κB signaling pathways?

When investigating PSTPIP2-NF-κB interactions:

• Temporal Resolution:

  • Implement time-course experiments (15 min, 30 min, 1h, 2h, 4h) following stimulation to capture dynamic interactions

  • Use synchronized cell populations to minimize heterogeneity in signaling responses

• Subcellular Fractionation:

  • Separate nuclear and cytoplasmic fractions to assess p65 nuclear translocation

  • Perform membrane fractionation to isolate PSTPIP2-containing compartments

• Proximity-Based Assays:

  • Implement proximity ligation assay (PLA) to visualize direct PSTPIP2-NF-κB interactions in situ

  • Consider FRET or BiFC approaches for living cell interaction studies

• Pathway Validation:

  • Use specific NF-κB inhibitors (PDTC at 25 μM) as positive controls

  • Implement CRISPR-based NF-κB reporter systems to quantify pathway activity

• Co-IP Optimization:

  • Crosslink samples prior to lysis to capture transient interactions

  • Use detergent conditions that preserve membrane-associated protein complexes (1% NP-40 or 0.5% CHAPS)

• Functional Validation:

  • Measure downstream target genes (IL-19, TNF-α) to confirm functional impact of interactions

  • Implement rescue experiments with constitutively active NF-κB constructs

These methodological considerations ensure robust characterization of PSTPIP2-NF-κB interactions.

How can emerging single-cell technologies be applied to study PSTPIP2 expression heterogeneity in inflammatory conditions?

Emerging single-cell technologies offer powerful approaches to characterize PSTPIP2 expression heterogeneity:

• Single-Cell RNA-Seq Integration:

  • Combine scRNA-seq with protein expression using CITE-seq to simultaneously measure PSTPIP2 mRNA and protein levels

  • Implement trajectory analysis to track PSTPIP2 expression changes during monocyte-to-macrophage differentiation

• Spatial Transcriptomics:

  • Apply Visium or Slide-seq to map PSTPIP2 expression within tissue microenvironments

  • Correlate spatial PSTPIP2 expression with inflammatory niches in synovial or kidney tissues

• CyTOF Applications:

  • Develop metal-conjugated PSTPIP2 antibodies for high-dimensional phenotyping

  • Create comprehensive immune cell panels that include PSTPIP2 alongside lineage and activation markers

• Live-Cell Imaging:

  • Implement lattice light-sheet microscopy with fluorescently-tagged PSTPIP2 to track dynamic changes in real-time

  • Apply optogenetic approaches to modulate PSTPIP2 function with spatiotemporal precision

• Multimodal Single-Cell Analysis:

  • Combine ATAC-seq with protein measurements to correlate chromatin accessibility with PSTPIP2 expression

  • Implement integrated single-cell multi-omics to capture epigenetic, transcriptomic, and proteomic regulation of PSTPIP2

These cutting-edge approaches will reveal unprecedented insights into cell-specific PSTPIP2 regulation in health and disease.

What are the most promising approaches for developing PSTPIP2-targeted therapeutics based on current antibody research?

Based on current antibody research, several promising approaches for PSTPIP2-targeted therapeutics include:

• Domain-Specific Modulation:

  • Develop antibodies targeting the F-BAR domain to modulate PSTPIP2 membrane localization

  • Design antibody-based therapeutics that stabilize PSTPIP2-PEST phosphatase interactions

• Cell-Specific Delivery Systems:

  • Engineer macrophage-targeting nanoparticles carrying PSTPIP2 expression constructs

  • Develop adeno-associated virus (AAV) vectors with tissue-specific promoters for localized PSTPIP2 expression

• Signaling Pathway Integration:

  • Target the PSTPIP2-NF-κB interaction interface with therapeutic antibodies or peptides

  • Develop bispecific antibodies linking PSTPIP2 with ERβ to enhance anti-inflammatory effects

• Conditional Activation Systems:

  • Implement small molecule-induced protein stabilization approaches for controlled PSTPIP2 upregulation

  • Develop proteolysis-targeting chimeras (PROTACs) to selectively degrade PSTPIP2 negative regulators

• Biomarker-Guided Therapies:

  • Establish PSTPIP2 expression profiles that predict response to anti-inflammatory biologics

  • Develop companion diagnostics using validated PSTPIP2 antibodies for patient stratification

These approaches represent promising avenues for translating PSTPIP2 research into therapeutic applications for inflammatory diseases.

What are the optimal fixation and permeabilization conditions for PSTPIP2 immunofluorescence staining in different cell types?

Optimal conditions vary by cell type and application:

For co-localization studies involving membrane structures, sequential staining protocols may yield superior results, with PSTPIP2 antibody incubation performed after cytoskeletal staining .

What are the critical parameters for quantitative Western blot analysis of PSTPIP2 in inflammatory disease models?

Critical parameters for quantitative Western blot analysis of PSTPIP2 include:

• Sample Preparation:

  • Use RIPA buffer supplemented with phosphatase inhibitors (10 mM NaF, 1 mM Na3VO4) and protease inhibitors

  • Maintain consistent protein extraction conditions across all experimental groups

  • Include centrifugation step (14,000g, 10 min, 4°C) to remove cell debris

• Protein Loading Controls:

  • Implement total protein normalization (TPN) using stain-free technology rather than single housekeeping proteins

  • Confirm consistent loading with Ponceau S staining prior to blocking

  • For phosphorylation studies, include both phospho-specific and total PSTPIP2 antibodies

• Electrophoresis Conditions:

  • Use gradient gels (4-15%) to optimize PSTPIP2 (36 kDa) separation

  • Include positive control lysates (RAW 264.7 cells) on each gel

  • Maintain consistent transfer conditions (25V, 1.3A, 10 min in Trans-Blot Turbo system)

• Quantification Parameters:

  • Establish linear dynamic range for PSTPIP2 antibody using serial dilutions

  • Capture images with 16-bit resolution within linear detection range

  • Use local background subtraction method for densitometry analysis

• Data Normalization Strategy:

  • Present data as fold-change relative to appropriate control conditions

  • For time-course experiments, normalize to time-matched controls

  • Include technical replicates (n=3 minimum) for statistical validity