PIK3R4 Antibody

What is PIK3R4 and what is its role in cellular pathways?

PIK3R4 (phosphoinositide-3-kinase regulatory subunit 4), also known as VPS15 or p150, is a 150 kDa adaptor protein that enhances the lipid kinase activity of PI3K complexes. It functions as a regulatory subunit of the PI3K complex that mediates formation of phosphatidylinositol 3-phosphate. Different complex forms play roles in multiple membrane trafficking pathways:

• The PI3KC3-C1 complex is involved in initiation of autophagosomes

• The PI3KC3-C2 complex contributes to maturation of autophagosomes and endocytosis

PIK3R4 is crucially involved in regulating membrane trafficking late in the endocytic pathway and autophagy . It contains 7 WD repeats, 3 HEAT repeats, and 1 protein kinase domain, and is crucial for VPS34 function and autophagosome formation in multicellular animals . Recent research has revealed an important role of PIK3R4 in cellular protection against ubiquitin-positive protein aggregates .

What applications are PIK3R4 antibodies validated for?

PIK3R4 antibodies have been validated for multiple applications with specific recommended dilutions:

Most PIK3R4 antibodies have been rigorously validated using positive controls such as human tonsil tissue, HeLa cells, and various cancer cell lines including TT human medullary thyroid cancer cells .

What is the difference between monoclonal and polyclonal PIK3R4 antibodies for research applications?

Polyclonal PIK3R4 Antibodies:

• Recognize multiple epitopes on the PIK3R4 protein

• Often provide stronger signals due to multiple binding sites

• Example: Anti-PIK3R4 polyclonal antibody (A06618) is raised against a 19 amino acid peptide near the carboxy terminus of human PIK3R4

• Generally good for applications requiring high sensitivity

• May have higher batch-to-batch variability

Monoclonal PIK3R4 Antibodies:

• Target a single epitope on the PIK3R4 protein

• Provide higher specificity for particular domains

• Example: Clone 1B5 (H00030849-M02) is raised against PIK3R4 (NP_055417, 1259 a.a. ~ 1358 a.a) partial recombinant protein

• Preferred for applications requiring consistent reproducibility

• More suitable for distinguishing specific isoforms or variants

Selection should be based on your specific experimental needs, including target region of interest and required specificity.

How can I validate the specificity of PIK3R4 antibodies for my experimental model?

A comprehensive validation strategy for PIK3R4 antibodies should include:

1. Western Blot Analysis:

• Confirm detection of a band at the expected molecular weight (153 kDa for full-length PIK3R4)

• Note that some antibodies may detect PIK3R4 at approximately 68 kDa , while others report 150 kDa

• Use appropriate positive controls (e.g., human tonsil tissue lysate, HeLa, TT, or MO7e cell lines)

2. Genetic Validation:

• Include PIK3R4 knockout or knockdown samples as negative controls

• Compare staining patterns before and after genetic manipulation

3. Peptide Competition Assay:

• Pre-incubate antibody with immunizing peptide before application

• Specific binding should be blocked by the peptide

• Some manufacturers offer blocking peptides specifically for this purpose

4. Cross-Reactivity Assessment:

• Test antibody against samples from multiple species if working with non-human models

• Verify sequence homology between species at the antibody epitope region

• Human PIK3R4 shares 96-99% amino acid sequence identity with mouse and rat orthologs

5. Multiple Antibody Approach:

• Compare results using antibodies targeting different epitopes of PIK3R4

• Consistent results across different antibodies suggest higher specificity

How does PIK3R4 expression correlate with immune infiltration in tumor microenvironments?

Recent research has revealed significant correlations between PIK3R4 expression and immune infiltration in cancer, particularly in diffuse large B-cell lymphoma (DLBCL):

• T-cell Infiltration:

  • High PIK3R4 expression is markedly related to CD8(+) T-cell infiltration (P < 0.05) in DLBCL patients

  • This suggests PIK3R4 may influence anti-tumor immunity through T-cell recruitment mechanisms

• Neutrophil Association:

  • PIK3R4 expression positively correlates with neutrophil infiltration in the tumor microenvironment (P < 0.05)

  • This correlation may impact tumor progression through inflammatory pathways

• ImmuneScore Correlation:

  • PIK3R4 level is significantly associated with the ImmuneScore in DLBCL (P < 0.05)

  • Interestingly, it was not linked to the ESTIMATEScore or StromalScore

• Immune Checkpoint Molecules:

  • PIK3R4 mRNA expression significantly correlates with several immune checkpoint molecules:

    • BTN3A2, BTN3A1 (butyrophilin family)

    • PRF1 (perforin)

    • CXCL9 (chemokine)

    • PDCD1 (PD-1)

    • TIGIT

  • These correlations suggest PIK3R4 may be a potential indicator of immunotherapy response

These findings indicate that PIK3R4 may function as a novel regulator of immune cell infiltration within tumor microenvironments, potentially serving as a valuable prognostic biomarker, particularly in DLBCL patients.

What are the optimal conditions for immunohistochemical detection of PIK3R4?

For optimal immunohistochemical detection of PIK3R4, researchers should consider:

Tissue Preparation:

• Formalin-fixed paraffin-embedded (FFPE) tissues have been successfully used

• 10% neutral buffered formalin fixation for 24-48 hours is recommended

• 4-5 μm section thickness is optimal for most applications

Antigen Retrieval:

• Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Pressure cooking for 3-5 minutes or microwave treatment for 10-20 minutes

Antibody Dilution and Incubation:

• Starting dilutions of 1:200 to 1:500 are recommended for most commercial antibodies

• For polyclonal antibodies like A06618, a concentration of 5 μg/ml has been validated

• Incubation at 4°C overnight generally provides optimal staining with minimal background

Detection Systems:

• HRP-conjugated polymer detection systems offer superior sensitivity

• DAB (3,3'-diaminobenzidine) is the most commonly used chromogen

• For multiplexing studies, consider fluorescent-based detection systems

Positive Control Selection:

• Human tonsil tissue has been validated as an excellent positive control

• Neuronal tissues may also show strong PIK3R4 expression

Counterstaining:

• Light hematoxylin counterstaining allows clear visualization of PIK3R4 immunoreactivity

• Avoid overstaining which can mask subtle immunoreactivity patterns

How can PIK3R4 be studied in the context of autophagy research?

PIK3R4 plays a crucial role in autophagy regulation, making it an important target for autophagy research:

1. PIK3R4-VPS34 Complex Analysis:

• PIK3R4 (also known as VPS15) forms a complex with VPS34 that is essential for autophagosome formation

• Co-immunoprecipitation assays can be used to study this interaction

• Recommended antibody dilution for immunoprecipitation: 1:50 to 1:100

2. Autophagy Flux Assays:

• Monitor LC3-II conversion in the presence and absence of autophagy inhibitors

• Assess PIK3R4's role by comparing wild-type and PIK3R4-depleted cells

• PIK3R4 knockdown should impair autophagosome formation

3. Live Cell Imaging:

• Use fluorescently tagged PIK3R4 to track its localization during autophagy induction

• Colocalization studies with other autophagy markers (LC3, WIPI2, ATG proteins)

• Time-lapse microscopy can reveal dynamic changes in PIK3R4 localization

4. PIK3R4 in Disease Models:

• PIK3R4 expression levels were significantly decreased at both pre-onset and onset stages of ALS disease in Tg(SOD1*G93A)1Gur mice

• This suggests a potential role in neurodegenerative disease pathogenesis

• Quantitative RT-PCR primers for PIK3R4 (forward: 5′-GAA CTT CAA GCA GCT CAT ACA AC-3′, reverse: 5′-GCT CAT GAA GAT GTG CAA CTAG-3′) can be used to assess expression levels

5. Phosphorylation Studies:

• PIK3R4 contains a protein kinase domain that may regulate autophagy

• Phospho-specific antibodies can help identify activation status

• Mass spectrometry approaches can identify novel phosphorylation sites

6. Membrane Trafficking Analysis:

• PIK3R4 regulates membrane trafficking in the endocytic pathway

• Vesicle tracking assays using fluorescent markers can reveal PIK3R4's role

• Electron microscopy remains the gold standard for examining autophagosome formation and structure

What are the prognostic implications of PIK3R4 expression in cancer research?

Recent studies have revealed significant prognostic implications of PIK3R4 expression in various cancers:

1. Diffuse Large B-cell Lymphoma (DLBCL):

• PIK3R4 mRNA levels in DLBCL tissues were significantly higher than in benign lymphadenitis tissues (P < 0.001)

• Median expression value: 21.52 (range 3.72-33.99) in tumor tissues vs. 11.03 (range 2.47-24.73) in benign tissues

• Diagnostic potential demonstrated by ROC curve analysis with AUC value of 0.861 (P < 0.001)

• High PIK3R4 expression associated with worse prognosis in DLBCL patients

2. Melanoma:

• Mutations in PIK3R4 have been found in metastatic melanoma

• Suggests potential role in cancer progression and metastasis

3. Ovarian Cancer:

• PIK3R4 gene copy number increases are associated with decreased survival in patients with ovarian tumors

• Indicates PIK3R4 may function as an oncogene in certain contexts

4. Research Applications:

• PIK3R4 expression analysis by qRT-PCR can be performed using published primer sets:

  • Forward: 5′-GAA CTT CAA GCA GCT CAT ACA AC-3′

  • Reverse: 5′-GCT CAT GAA GAT GTG CAA CTAG-3′

• Immunohistochemical scoring of PIK3R4 protein expression can provide additional prognostic information

• Combined analysis of PIK3R4 with immune cell markers may offer enhanced prognostic value

These findings suggest that PIK3R4 could serve as a novel diagnostic, prognostic, or therapeutic biomarker in multiple cancer types, particularly in DLBCL.

How can I troubleshoot non-specific staining or high background when using PIK3R4 antibodies?

Non-specific staining and high background are common challenges when working with PIK3R4 antibodies. Here are methodological approaches to address these issues:

For Western Blotting:

• Blocking Optimization:

  • Test different blocking agents (5% non-fat milk, 5% BSA, commercial blockers)

  • Increase blocking time to 2 hours at room temperature

  • For PIK3R4 detection, 5% BSA often provides better results than milk-based blockers

• Antibody Dilution Adjustment:

  • Start with manufacturer's recommended dilution (typically 1-2 μg/ml for PIK3R4)

  • Prepare a dilution series if optimal concentration is unknown

  • Higher dilutions (lower antibody concentration) often reduce background

• Washing Protocol Enhancement:

  • Increase wash volume and duration (5 x 5 minutes with TBST)

  • Use fresh washing buffer for each wash step

  • Add 0.1-0.3% Tween-20 to reduce hydrophobic interactions

For Immunohistochemistry/Immunofluorescence:

• Endogenous Peroxidase Quenching:

  • Treat sections with 3% hydrogen peroxide for 10 minutes

  • For fluorescence applications, use commercial background reducers

• Antigen Retrieval Optimization:

  • Compare citrate buffer (pH 6.0) vs. EDTA buffer (pH 9.0)

  • Adjust retrieval time and temperature

  • For PIK3R4, EDTA-based retrieval often yields cleaner results

• Antibody Incubation Conditions:

  • Reduce primary antibody concentration (start at 5 μg/ml for IHC-P)

  • Incubate at 4°C overnight rather than at room temperature

  • Add 0.1% Triton X-100 to reduce non-specific binding

• Negative Controls:

  • Include isotype control antibody (IgG2a for monoclonal)

  • Omit primary antibody but include all other steps

  • Pre-absorb antibody with immunizing peptide when available

What are the critical factors to consider when selecting a PIK3R4 antibody for specific research applications?

When selecting a PIK3R4 antibody for your research, consider these critical factors:

1. Target Epitope Location:

• Antibodies targeting different regions of PIK3R4 may yield different results

• N-terminal epitopes: Better for detecting full-length protein

• C-terminal epitopes: May detect truncated forms or specific domains

• Example: Some PIK3R4 antibodies target a 19 amino acid peptide near the carboxy terminus (amino acids 1290-1340) , while others target amino acids 1259-1358

2. Validated Applications:

• Ensure the antibody has been validated for your specific application

• Cross-check validation images in product datasheets

• Example applications with validated dilutions:

  • Western blot: 1:500-1:1000

  • IHC-P: 1:200-1:500 (5 μg/ml)

  • IF: 20 μg/ml

3. Species Reactivity:

• Confirm reactivity with your experimental species

• Human PIK3R4 shares high sequence homology with mouse (98%) and rat (98%)

• Some antibodies are validated for multiple species (human, mouse, rat, monkey)

4. Clone Type and Format:

• Monoclonal antibodies (e.g., clone 1B5 ) offer high reproducibility

• Polyclonal antibodies may provide higher sensitivity for low abundance targets

• Consider special formats needed (BSA-free, azide-free) for specific applications

5. Publication Record:

• Check if the antibody has been cited in peer-reviewed publications

• Look for successful use in applications similar to yours

• Examine supplementary methods in publications for optimization tips

6. Molecular Weight Detection:

• Some antibodies detect PIK3R4 at ~150 kDa (calculated MW: 153 kDa)

• Others report detection at ~68 kDa

• Verify which form of PIK3R4 is relevant to your research question

How can I optimize immunofluorescence protocols for detecting PIK3R4 in different subcellular compartments?

PIK3R4 localizes to multiple subcellular compartments, including late endosomes, autophagosomes, and the microtubule cytoskeleton . Optimizing immunofluorescence protocols for specific compartments requires:

1. Fixation Method Selection:

• For membrane-associated PIK3R4:

  • 4% paraformaldehyde (10-15 minutes at room temperature)

  • Avoid methanol fixation which can disrupt membrane structures

• For cytoskeletal-associated PIK3R4:

  • Combined fixation: 4% PFA followed by brief methanol treatment (-20°C, 5 minutes)

  • Preserves both membrane and cytoskeletal structures

2. Permeabilization Optimization:

• For cytoplasmic and membrane signals:

  • 0.1-0.2% Triton X-100 (5-10 minutes)

• For endosomal compartments:

  • Mild permeabilization with 0.05% saponin

  • Maintains endosomal integrity while allowing antibody access

3. Co-localization Markers:

• Late endosomes: Co-stain with Rab7 or LAMP1

• Autophagosomes: Co-stain with LC3 or ATG proteins

• Golgi-associated PIK3R4: Co-stain with GM130

• Cytoskeletal PIK3R4: Co-stain with α-tubulin

4. Signal Amplification Strategies:

• Tyramide signal amplification for detecting low abundance PIK3R4

• Use of high-sensitivity detection systems (e.g., Alexa Fluor 647-conjugated secondary antibodies)

• Recommended primary antibody concentration: 20 μg/ml

5. Confocal Microscopy Settings:

• Z-stack acquisition to capture the 3D distribution of PIK3R4

• Appropriate pinhole settings (1 Airy unit) for optimal resolution

• Sequential scanning to prevent bleed-through when performing co-localization studies

6. Image Analysis Approaches:

• Quantify co-localization using Pearson's or Manders' coefficients

• Measure fluorescence intensity in specific compartments

• Compare signal distribution in control vs. experimental conditions

Validated cell lines for PIK3R4 immunofluorescence include HeLa cells, where clear subcellular distribution patterns have been documented .

How is PIK3R4 being explored as a potential biomarker in disease research?

PIK3R4 is emerging as a promising biomarker in several disease contexts:

1. Diffuse Large B-cell Lymphoma (DLBCL):

• Significantly higher expression in DLBCL tissues compared to benign lymphadenitis (P<0.001)

• ROC curve analysis showed high diagnostic value (AUC = 0.861, P<0.001)

• Potentially useful for distinguishing DLBCL from benign lymphoid proliferation

2. Neurodegenerative Disorders:

• PIK3R4 expression significantly decreased in ALS models (Tg(SOD1*G93A)1Gur mice)

• Detected at both pre-onset and onset stages of disease

• Associated with Neurodegeneration With Brain Iron Accumulation

3. Myopathy Research:

• Associated with X-Linked Myopathy With Excessive Autophagy

• Suggests potential role in muscle disorders related to autophagy dysregulation

4. Cancer Progression:

• PIK3R4 mutations found in metastatic melanoma

• Copy number increases associated with decreased survival in ovarian cancer patients

• May serve as a prognostic indicator for patient stratification

5. Immune Response Monitoring:

• Significant correlation with immune cell infiltration (CD8+ T-cells, neutrophils)

• Association with multiple immune checkpoint molecules (BTN3A2, BTN3A1, PRF1, CXCL9, PDCD1, TIGIT)

• Potential predictor of immunotherapy response

Methodological Considerations for Biomarker Studies:

• RNA-based detection: qRT-PCR using validated primers

• Protein-based detection: IHC scoring systems based on staining intensity and distribution

• Combined approaches incorporating PIK3R4 with other markers may provide enhanced predictive value

What are the latest findings regarding PIK3R4's role in autophagy and cell signaling pathways?

Recent research has expanded our understanding of PIK3R4's role in autophagy and signaling:

1. Autophagy Regulation:

• PIK3R4/VPS15 is crucial for VPS34 function and autophagosome formation

• Essential component of the PI3KC3-C1 complex involved in autophagy initiation

• Critical for the PI3KC3-C2 complex's role in autophagosome maturation

• Cellular protection against ubiquitin-positive protein aggregates depends on PIK3R4

2. Membrane Trafficking Regulation:

• Regulates degradative endocytic trafficking pathways

• Involved in the trafficking of mATG9 from the Golgi to phagophores and autophagosomes

• May be recruited to autophagosomes in a SLAM-dependent manner following exposure to gram-negative bacteria

3. Cytokinesis Control:

• PIK3R4 participates in regulation of cytokinesis

• Likely functions in this process as part of the PI3KC3-C2 complex

• Suggests role in cell division beyond its canonical autophagy functions

4. Signaling Pathway Connections:

• Associated with Prolactin Signaling pathway

• Involved in Apoptotic Pathways in Synovial Fibroblasts

• Functions upstream of several key cellular processes:

  • DNA damage response

  • rRNA metabolic process

  • Mitochondrial gene expression

  • Nucleoside metabolic process

5. Protein Interactions:

• Contains 7 WD repeats, 3 HEAT repeats, and 1 protein kinase domain

• WD repeats mediate protein-protein interactions

• Kinase domain suggests potential signaling roles beyond structural functions

• Myristoylation facilitates membrane association and regulates activity of hVps34