PIKFYVE Antibody

What is PIKFYVE and why is it significant in cellular research?

PIKFYVE is a lipid kinase approximately 237-240 kDa in size that plays critical roles in lysosomal function, autophagy, and endosomal trafficking . The gene is embryonically lethal when knocked out, indicating its vital role in early development . Recent research has identified PIKFYVE as integral to lysosomal functioning and as a novel targetable vulnerability in pancreatic ductal adenocarcinoma (PDAC) . It's particularly significant because PIKFYVE inhibition disrupts lysosome function in autophagy and can selectively kill certain cancer cells, as well as potentiate immune response to checkpoint blockade therapy .

What applications are PIKFYVE antibodies most commonly used for?

PIKFYVE antibodies are predominantly used for Western blotting, immunohistochemistry, and ELISA applications in research settings . The Human PIKFyve Antibody (AF7885) has been validated for Western blotting, showing specific detection of PIKFyve at approximately 240 kDa in Jurkat and K562 human cell lines . Similarly, the PIKFYVE (E4X3R) Rabbit mAb (#92839) has been validated for Western blotting applications with a recommended dilution of 1:1000 . These antibodies are crucial tools for studying PIKFYVE expression levels, localization, and function in various cellular processes.

How should PIKFYVE antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of PIKFYVE antibodies are essential for maintaining their activity and specificity. Based on manufacturer recommendations:

• Store unopened antibodies at -20 to -70°C for up to 12 months from the date of receipt

• Once reconstituted, store at 2 to 8°C under sterile conditions for up to 1 month

• For longer-term storage after reconstitution, aliquot and store at -20 to -70°C for up to 6 months

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles as these significantly reduce antibody activity

• Some antibodies, like the PIKFYVE (E4X3R) Rabbit mAb, should not be aliquoted to maintain consistency

Adhering to these storage guidelines ensures maximum antibody performance and reproducibility in experimental applications.

How can I validate the specificity of a PIKFYVE antibody for my experimental system?

Validating antibody specificity is critical for reliable research outcomes. For PIKFYVE antibodies, consider these methodological approaches:

• Positive controls: Use cell lines known to express PIKFYVE, such as Jurkat human acute T cell leukemia and K562 human chronic myelogenous leukemia cell lines, which have been validated for Human PIKFyve Antibody (AF7885)

• Knockout/knockdown validation: Compare antibody signals between wild-type and PIKFYVE-knockout or knockdown samples. The search results mention PIKFYVE knockdown in MIA PaCa-2 and PANC-1 human PDAC cell lines using two independent sgRNAs, which could serve as negative controls

• Band size verification: Confirm detection at the expected molecular weight of approximately 240 kDa

• Cross-reactivity assessment: Check species cross-reactivity—the PIKFYVE (E4X3R) Rabbit mAb has been tested for human and mouse reactivity

• Functional validation: After PIKFYVE knockdown or inhibition, verify expected phenotypes such as increased LC3A/B-II to LC3A/B-I ratio, increased p62 levels (indicating inhibition of autophagic flux), and lysosomal vacuolization

What experimental conditions are optimal for detecting PIKFYVE by Western blot?

Based on the provided search results, optimal Western blot conditions for PIKFYVE detection include:

• Sample preparation: Use cell lysates like those from Jurkat or K562 human cell lines

• Membrane selection: PVDF membranes have been successfully used for PIKFYVE detection

• Antibody dilution: Use 0.25 μg/mL for Human PIKFyve Antigen Affinity-purified Polyclonal Antibody (Catalog # AF7885) or 1:1000 dilution for PIKFYVE (E4X3R) Rabbit mAb

• Secondary antibody: Follow with appropriate HRP-conjugated secondary antibody, such as Anti-Sheep IgG Secondary Antibody for AF7885

• Reducing conditions: PIKFYVE detection has been successful under reducing conditions

• Buffer systems: Use appropriate immunoblot buffer groups (e.g., Immunoblot Buffer Group 1 has been validated)

Expect to detect a specific band for PIKFYVE at approximately 240 kDa when using these conditions.

How does PIKFYVE inhibition affect autophagic flux, and what markers should I monitor?

PIKFYVE inhibition has significant and well-characterized effects on autophagic flux that can be monitored through specific markers:

• LC3 conversion: PIKFYVE knockdown increases the LC3A/B-II to LC3A/B-I ratio, indicating disruption of autophagy

• p62/SQSTM1 accumulation: Increased p62 levels are observed following PIKFYVE inhibition, confirming inhibition of autophagic flux

• Lysosomal morphology: Both genetic knockdown of PIKFYVE and pharmacological inhibition with compounds like apilimod or ESK981 induce a characteristic lysosomal vacuolization phenotype visible within four hours of treatment

• Experimental approaches: Western blotting for LC3 and p62, combined with microscopic analysis of lysosomal morphology, provides a comprehensive assessment of autophagic flux disruption

These markers should be monitored in time-course experiments, as the lysosomal vacuolization phenotype appears rapidly (within four hours) while the metabolic consequences and cell viability effects may take longer to manifest .

What are the comparative advantages of genetic versus pharmacological PIKFYVE inhibition in cancer models?

Both genetic and pharmacological approaches to PIKFYVE inhibition have distinct advantages in cancer research:

Genetic Inhibition Advantages:

• Specificity: CRISPR/Cas9-mediated knockdown provides high target specificity

• Complete ablation: Can achieve complete loss of PIKFYVE function

• Long-term studies: Enables the study of long-term consequences of PIKFYVE loss

• Cell-type specificity: Conditional knockout models allow for tissue-specific PIKFYVE deletion

Pharmacological Inhibition Advantages:

• Clinical relevance: Compounds like apilimod and ESK981 have cleared phase 1 clinical trials

• Dosage control: Allows for titration and reversible inhibition

• Temporal control: Can inhibit PIKFYVE at specific timepoints during experiments

• Combinatorial studies: Easier to combine with other treatments

Methodological Considerations:

• Researchers have used a genetically engineered mouse model (GEMM) with conditional deletion of Pikfyve to study its role in PDAC, which dramatically increased animal survival and decreased disease burden

• Prophylactic pharmacological inhibition of PIKfyve with ESK981 also decreased PDAC disease burden in a GEMM of PDAC

• Both approaches revealed that PIKFYVE inhibition substantially slows the growth of PDAC cells, with pharmacological inhibitors showing IC50 values in the nanomolar range

The method selection should be guided by the specific research question, with genetic approaches favored for mechanistic studies and pharmacological approaches for therapeutic potential assessment.

How can I design experiments to study the role of PIKFYVE in immune checkpoint blockade therapy response?

Designing experiments to study PIKFYVE's role in immune checkpoint blockade (ICB) therapy response requires a multi-faceted approach:

• Single-cell RNA-seq analysis:

  • Analyze PIKFYVE expression in immune cells across multiple cancer types

  • Compare PIKFYVE expression in conventional dendritic cells (cDC) between ICB responders and non-responders

  • Focus on cell-type specific expression patterns, as PIKFYVE expression in DCs, but not other immune cells, correlates with ICB response

• In vivo experimental design:

  • Use genetic models with CD11c-specific Pikfyve deletion to study DC-mediated immune responses

  • Design combination therapy experiments with PIKfyve inhibitors (like apilimod) and ICB agents

  • Include vaccine adjuvants in treatment strategies to assess synergistic effects

• Functional assays:

  • Measure antigen presentation using H2-kb-SIINFEKL surface expression on cDCs

  • Assess DC-dependent T cell immunity through T cell activation markers and proliferation assays

  • Monitor tumor growth and immune infiltration in response to PIKfyve inhibition and ICB combination

• Mechanistic investigations:

  • Study how PIKfyve ablation affects the non-canonical NF-κB pathway in CD11c+ cells

  • Investigate changes in antigen processing and presentation pathways

  • Analyze changes in cytokine production and immune cell recruitment

This experimental approach will provide comprehensive insights into how PIKFYVE modulates immune response to ICB therapy and potential therapeutic strategies.

What methods are most effective for studying PIKFYVE-dependent lipid metabolism in cancer cells?

Studying PIKFYVE-dependent lipid metabolism in cancer cells requires specialized methodologies:

• CRISPR-based metabolic screening:

  • Implement metabolism-focused CRISPR screens to identify synthetic dependencies with PIKFYVE inhibition

  • Research has revealed synthetic dependency on de novo fatty acid synthesis genes (e.g., FASN and ACACA) when PIKFYVE is inhibited in PDAC

• Thermal shift assays:

  • Use Cellular Thermal Shift Assay (CETSA) to confirm binding of PIKFYVE inhibitors to the target protein in cells

  • This method helps validate the on-target effects of inhibitors like apilimod and ESK981

• Functional assays for metabolic dependencies:

  • Assess cell viability with PIKfyve inhibitors across multiple concentrations to determine IC50 values

  • Compare sensitivity profiles across different cancer cell lines

  • Combine PIKFYVE inhibition with inhibitors of fatty acid synthesis to confirm synthetic lethality

• In vivo metabolic studies:

  • Use prophylactic treatment with PIKFYVE inhibitors in genetically engineered mouse models to assess impact on tumor metabolism

  • Measure changes in pancreata weight and compare to wild-type controls

• Subcellular fractionation and lipid analysis:

  • Isolate lysosomes to study PIKFYVE-dependent phosphoinositide conversion

  • Employ lipidomics approaches to quantify changes in phosphatidylinositol species

These methodologies provide comprehensive insights into how PIKFYVE regulates lipid homeostasis in cancer cells and can help identify novel therapeutic vulnerabilities.

How can I optimize immunofluorescence protocols to study PIKFYVE co-localization with autophagy markers?

Optimizing immunofluorescence protocols for PIKFYVE co-localization with autophagy markers requires careful consideration of several technical aspects:

• Fixation methods:

  • Use 4% paraformaldehyde for 15-20 minutes at room temperature to preserve membrane structures

  • Avoid methanol fixation which can disrupt membrane-associated proteins like PIKFYVE

• Antibody selection and validation:

  • Use antibodies validated for immunofluorescence applications

  • Confirm specificity using PIKFYVE knockout or knockdown cells

  • For autophagy markers, select antibodies against LC3B, p62/SQSTM1, and LAMP1/LAMP2

• Detection of vacuolization phenotype:

  • Following PIKFYVE inhibition, lysosomal vacuolization occurs within four hours

  • Use this phenotype as a positive control for successful PIKFYVE inhibition

  • Combine with LC3 and p62 staining to correlate vacuolization with autophagy disruption

• Live cell imaging considerations:

  • For dynamic studies, consider fluorescently-tagged PIKFYVE constructs

  • Monitor real-time changes in response to inhibitors like apilimod or ESK981

  • Use acidotropic dyes like LysoTracker to visualize enlargement of acidic compartments

• Quantification approaches:

  • Measure vacuole size, number, and distribution

  • Quantify co-localization using Pearson's or Mander's correlation coefficients

  • Analyze changes in autophagy marker distribution and intensity

These optimized protocols will enable detailed analysis of how PIKFYVE inhibition affects the spatial organization of the autophagy-lysosomal system and provide valuable insights into the mechanisms of PIKFYVE-dependent cell death in cancer models.

What controls should I include when using PIKFYVE antibodies in my experiments?

When designing experiments with PIKFYVE antibodies, include these essential controls:

• Positive controls:

  • Cell lines with known PIKFYVE expression: Jurkat human acute T cell leukemia and K562 human chronic myelogenous leukemia cell lines

  • Human PDAC cell lines: MIA PaCa-2, PANC-1, Panc 04.03, and murine KPC (7940B) cells

• Negative controls:

  • PIKFYVE knockout or knockdown samples using CRISPR/Cas9 or siRNA

  • Blocking peptide controls to confirm antibody specificity

  • Secondary antibody-only controls to assess non-specific binding

• Validation controls:

  • Cross-validation with multiple PIKFYVE antibodies targeting different epitopes

  • Functional validation: Following PIKFYVE inhibition, confirm expected phenotypes (increased LC3-II/LC3-I ratio, p62 accumulation, and lysosomal vacuolization)

• Loading controls:

  • For Western blotting, use housekeeping proteins appropriate for your experimental system

  • Note that PIKFYVE is a high molecular weight protein (~240 kDa), so verify complete transfer

Including these controls will ensure the reliability and reproducibility of your PIKFYVE antibody-based experiments.

What are the common technical challenges when working with PIKFYVE antibodies and how can they be addressed?

PIKFYVE antibodies present several technical challenges that can be addressed with specific methodological approaches:

• High molecular weight detection issues:

  • Challenge: PIKFYVE's large size (~240 kDa) can make detection difficult

  • Solution: Use gradient gels (4-15%), extend transfer time, and verify complete transfer with high molecular weight markers

• Low expression levels:

  • Challenge: Endogenous PIKFYVE may be expressed at low levels in some cell types

  • Solution: Optimize protein loading, increase antibody concentration, and use enhanced chemiluminescence detection systems with longer exposure times

• Non-specific binding:

  • Challenge: Some antibodies may show cross-reactivity with other proteins

  • Solution: Increase blocking time/concentration, optimize antibody dilution, and validate with PIKFYVE knockdown controls

• Batch-to-batch variability:

  • Challenge: Different lots of the same antibody may perform differently

  • Solution: Consider recombinant antibodies like PIKFYVE (E4X3R) Rabbit mAb which offers superior lot-to-lot consistency

• Tissue-specific optimization:

  • Challenge: Different tissue types may require modified protocols

  • Solution: Adjust fixation methods, antigen retrieval conditions, and antibody incubation times based on tissue type

Addressing these challenges will improve the reliability and reproducibility of experiments using PIKFYVE antibodies.

How do I interpret changes in PIKFYVE expression levels across different cancer types?

Interpreting PIKFYVE expression data across cancer types requires consideration of several factors:

• Baseline expression patterns:

  • PIKFYVE is expressed in multiple cell types across various cancers

  • Single-cell RNA-seq analysis reveals cell type-specific expression patterns

  • Cancer cells in PDAC models show higher PIKFYVE transcripts than surrounding normal pancreatic cells

• Correlation with clinical outcomes:

  • Lower PIKFYVE expression in conventional DCs (cDCs) correlates with better response to immune checkpoint blockade (ICB) therapy in melanoma patients

  • A similar pattern was observed in a patient with endometrial cancer who had complete response to therapy

  • No correlation was found between PIKFYVE expression in other immune cell types and ICB response

• Functional implications:

  • High PIKFYVE expression may indicate dependence on PIKfyve-driven lipid metabolism

  • Cancer cells with elevated PIKFYVE expression may be more sensitive to PIKfyve inhibitors

  • In immune cells, particularly DCs, PIKFYVE expression negatively regulates cell function and anti-tumor immunity

• Analytical approaches:

  • Use appropriate normalization methods when comparing expression across datasets

  • Consider cell type-specific expression patterns rather than bulk tissue analysis

  • Validate RNA expression findings with protein-level analysis using PIKFYVE antibodies

This nuanced interpretation of PIKFYVE expression data can guide the development of personalized therapeutic strategies targeting this pathway.

What is the significance of PIKFYVE antibody staining patterns in cellular compartments?

The subcellular localization pattern of PIKFYVE revealed by antibody staining provides important functional insights:

• Normal localization patterns:

  • PIKFYVE primarily localizes to endosomal compartments

  • It contains a FYVE domain that binds specifically to phosphatidylinositol 3-phosphate (PI3P) on endosomal membranes

  • The protein plays a role in converting PI3P to phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2)

• Changes following inhibition or perturbation:

  • PIKFYVE inhibition causes characteristic lysosomal vacuolization visible within four hours of treatment

  • This vacuolization phenotype serves as a cellular biomarker for successful PIKFYVE inhibition

  • The disruption of normal PIKFYVE localization correlates with inhibition of autophagic flux

• Co-localization significance:

  • Co-localization with LC3 and p62/SQSTM1 indicates involvement in autophagosome-lysosome fusion

  • Association with endosomal markers reflects PIKFYVE's role in endosomal trafficking and maturation

  • Reduced co-localization with lysosomes following inhibitor treatment suggests functional impairment

• Interpretation guidelines:

  • Punctate staining patterns are typical for endosomal/lysosomal proteins

  • Diffuse cytoplasmic staining may indicate antibody non-specificity or protein overexpression

  • Changes in localization pattern following treatment can indicate functional responses

Understanding these staining patterns helps researchers interpret the functional consequences of PIKFYVE modulation in their experimental systems.

How can PIKFYVE antibodies be used to identify potential responders to PIKFYVE inhibitor therapy?

PIKFYVE antibodies can serve as valuable biomarker tools to identify potential responders to PIKFYVE inhibitor therapy:

• Profiling PIKFYVE expression in patient samples:

  • Use validated PIKFYVE antibodies for immunohistochemical analysis of tumor biopsies

  • Quantify PIKFYVE expression levels in both tumor cells and tumor-infiltrating immune cells

  • Focus particularly on PIKFYVE expression in conventional dendritic cells within the tumor microenvironment

• Predictive biomarker development:

  • Patients with higher PIKFYVE expression in tumor cells may be more susceptible to direct anti-tumor effects of PIKFYVE inhibitors

  • Conversely, patients with high PIKFYVE expression in conventional DCs may benefit most from immune-modulatory effects of PIKFYVE inhibition

  • Single-cell analysis of patient samples can help identify cell type-specific expression patterns that predict response

• Functional assays with patient-derived materials:

  • Treat patient-derived organoids or xenografts with PIKFYVE inhibitors ex vivo

  • Use PIKFYVE antibodies to confirm target engagement and pathway modulation

  • Correlate functional responses with baseline PIKFYVE expression patterns

• Companion diagnostic potential:

  • PIKFYVE antibody-based assays could be developed as companion diagnostics for PIKFYVE inhibitor clinical trials

  • Standardized immunohistochemistry protocols would need to be developed and validated

  • Cut-off values for "high" versus "low" PIKFYVE expression would need to be established through clinical correlation studies

This biomarker-driven approach could significantly enhance patient selection for PIKFYVE inhibitor therapies and improve clinical outcomes.

What is the role of PIKFYVE in cancer immunotherapy resistance mechanisms?

PIKFYVE plays a significant role in cancer immunotherapy resistance through several mechanisms:

• Modulation of dendritic cell function:

  • High PIKFYVE expression in conventional DCs is associated with poor patient response to immune checkpoint blockade (ICB) therapy

  • PIKFYVE negatively regulates DC function through selective alteration of the non-canonical NF-κB pathway

  • Inhibition of PIKFYVE enhances DC-dependent T cell immunity

• Impact on tumor microenvironment:

  • PIKFYVE inhibition can enhance the function of CD11c+ cells (predominantly dendritic cells)

  • Both genetic deletion of Pikfyve in CD11c+ cells and pharmacological inhibition with apilimod restrain tumor growth

  • This effect potentiates ICB efficacy in tumor-bearing mouse models

• Combination therapy opportunities:

  • PIKFYVE inhibitors like apilimod can be combined with vaccine adjuvants to reduce tumor progression

  • This combination approach represents a promising strategy for cancer immunotherapy

  • The synergistic effect likely occurs through enhanced antigen presentation and T cell activation

• Translational implications:

  • Patients with high PIKFYVE expression in DCs might benefit from combination therapy with PIKFYVE inhibitors and ICB

  • Monitoring PIKFYVE expression in tumor-infiltrating DCs could serve as a biomarker for immunotherapy resistance

  • Targeting DC-specific PIKFYVE activity may overcome resistance mechanisms in tumors unresponsive to standard immunotherapies

Understanding PIKFYVE's role in immunotherapy resistance opens new avenues for developing more effective cancer treatment strategies.