INPP5F Antibody, HRP conjugated

What is INPP5F and what cellular functions does it regulate?

INPP5F (Inositol polyphosphate 5-phosphatase F) is primarily an inositol 4-phosphatase that acts on phosphatidylinositol 4-phosphate. It plays crucial roles in endocytic recycling through sequential dephosphorylation of phosphatidylinositol 4,5-bisphosphate. INPP5F regulates several important signaling pathways including the TF:TFRC and integrins recycling pathway, modulates the AKT/GSK3B pathway by decreasing AKT and GSK3B phosphorylation, and negatively regulates STAT3 signaling through inhibition of STAT3 phosphorylation and nuclear translocation. Additionally, it functions as an important modulator of cardiac myocyte size and stress response, and may negatively regulate axon regeneration after central nervous system injuries .

How does the HRP conjugation affect the application range of INPP5F antibodies?

The HRP (horseradish peroxidase) conjugation to INPP5F antibodies expands their application potential, particularly for ELISA-based detection systems. While unconjugated antibodies require secondary detection reagents, the direct HRP conjugation allows for immediate enzymatic reaction with appropriate substrates, streamlining detection workflows. The HRP conjugation makes these antibodies especially suitable for highly sensitive detection in ELISA applications, while maintaining their specificity to the target epitope (amino acids 187-204 of human INPP5F protein) . The conjugation provides significant advantages in terms of reducing background signal and cross-reactivity that can occur with two-antibody detection systems.

What is the functional relationship between INPP5F and OCRL in phosphoinositide metabolism?

INPP5F is functionally linked to OCRL in phosphoinositide metabolism, creating a coordinated system for sequential dephosphorylation of phosphatidylinositol 4,5-bisphosphate. OCRL catalyzes the hydrolysis of the 5-position phosphate of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), converting it to phosphatidylinositol 4-phosphate . INPP5F then acts on this product by removing the 4-position phosphate. This sequential action at the 5 and 4 positions of inositol plays a critical role in regulating endocytic recycling pathways and membrane trafficking. The functional relationship between these two phosphatases represents an important mechanism for controlling phosphoinositide dynamics in cellular compartments, particularly in endosomal trafficking .

What are the optimal conditions for using INPP5F antibody, HRP conjugated in ELISA assays?

For optimal ELISA performance with INPP5F antibody, HRP conjugated, researchers should consider the following protocol:

• Coating: Coat ELISA plates with target antigen at 1-10 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C

• Blocking: Block with 3-5% BSA or non-fat milk in PBS-T (PBS with 0.05% Tween-20) for 1-2 hours at room temperature

• Primary antibody: Dilute the HRP-conjugated INPP5F antibody appropriately (starting with manufacturer's recommendations, typically 1:1000 to 1:5000) in blocking buffer

• Incubation: Apply diluted antibody to wells and incubate for 1-2 hours at room temperature

• Washing: Wash 4-5 times with PBS-T to remove unbound antibody

• Detection: Add TMB or other HRP substrate and incubate until color develops (typically 5-30 minutes)

• Stopping reaction: Add stop solution (usually 2N H₂SO₄)

• Measurement: Read absorbance at appropriate wavelength (450nm for TMB)

The antibody has been validated specifically for human INPP5F detection, with the immunogen being a peptide sequence spanning amino acids 187-204 of human Phosphatidylinositide phosphatase SAC2 protein .

How can researchers effectively use INPP5F antibody for immunofluorescence studies?

For effective immunofluorescence studies using INPP5F antibody:

• Sample preparation:

  • Cell fixation: Use 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilization: 0.1-0.5% Triton X-100 in PBS for 10 minutes

  • Blocking: 5% normal serum (from the species of secondary antibody) with 1% BSA for 1 hour

• Primary antibody application:

  • For unconjugated INPP5F antibody: Dilute 1:100-1:500 in blocking buffer and incubate overnight at 4°C

  • For direct detection methods: Use FITC-conjugated INPP5F antibody (available as alternative to HRP-conjugated)

• Secondary antibody (if using unconjugated primary):

  • Use appropriate species-specific fluorescent-labeled secondary antibody

  • Incubate for 1-2 hours at room temperature in the dark

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI (1:1000) for 5 minutes

  • Mount with anti-fade mounting medium

• Imaging considerations:

  • INPP5F typically shows cytoplasmic localization with potential enrichment in endosomal compartments

  • Co-staining with endosomal markers (e.g., EEA1, Rab5) can provide insight into INPP5F functional localization

This approach can effectively visualize INPP5F's subcellular distribution and potentially its colocalization with interacting partners like OCRL .

What methods can be used to validate INPP5F antibody specificity in experimental systems?

To validate INPP5F antibody specificity, researchers should employ multiple complementary approaches:

• Western blot analysis:

  • Comparing signal from wild-type samples versus INPP5F knockdown/knockout samples

  • Expected molecular weight of human INPP5F is approximately 126 kDa

  • Testing multiple cell lines with known INPP5F expression levels

• Peptide competition assay:

  • Pre-incubating the antibody with excess immunizing peptide (amino acids 187-204 of human INPP5F)

  • Comparing results with and without peptide blocking

  • Signal should be significantly reduced or eliminated when the specific peptide is present

• Immunoprecipitation followed by mass spectrometry:

  • Confirming that the antibody pulls down INPP5F protein

  • Identifying any potential cross-reactive proteins

• Orthogonal detection methods:

  • Comparing results with alternative INPP5F antibodies recognizing different epitopes

  • Correlating protein detection with mRNA expression (RT-qPCR)

• Recombinant expression:

  • Overexpressing tagged INPP5F and detecting with both tag-specific and INPP5F antibodies

These validation steps are critical for ensuring experimental rigor, particularly in studies examining INPP5F's role in signaling pathways and cardiac function .

How can researchers investigate the interaction between INPP5F and the AKT/GSK3B signaling pathway?

To investigate INPP5F's modulation of the AKT/GSK3B pathway, researchers should consider a multi-faceted approach:

• Phosphorylation analysis:

  • Western blot analysis of phospho-AKT (Ser473 and Thr308) and phospho-GSK3B (Ser9) levels following INPP5F manipulation

  • Comparison between control, INPP5F overexpression, and INPP5F knockdown conditions

  • Time-course analysis after pathway stimulation (e.g., insulin, growth factors)

• Functional assays:

  • Establish cell models with INPP5F overexpression or knockdown

  • Measure downstream functional outcomes of AKT/GSK3B signaling (e.g., glycogen synthesis, cell survival)

  • Use pathway-specific inhibitors (e.g., PI3K inhibitors) to confirm specificity

• Phosphoinositide quantification:

  • Measure PIP₂ and PIP₃ levels using biochemical assays or fluorescent biosensors

  • Correlate INPP5F activity with phosphoinositide dynamics and AKT membrane recruitment

• Co-immunoprecipitation:

  • Investigate physical interactions between INPP5F and AKT/GSK3B pathway components

  • Use the INPP5F antibody to pull down protein complexes

• Subcellular localization:

  • Perform immunofluorescence co-localization studies of INPP5F with AKT and GSK3B

  • Analyze translocation dynamics following pathway activation

This comprehensive approach can elucidate how INPP5F decreases AKT and GSK3B phosphorylation, as mentioned in the protein background information .

What are the considerations for studying INPP5F's role in cardiac stress response using antibody-based techniques?

Studying INPP5F's role in cardiac stress requires careful experimental design:

• Model selection and validation:

  • Choose appropriate cardiac models (primary cardiomyocytes, cardiac cell lines, or animal models)

  • Validate INPP5F antibody specificity in cardiac tissue specifically

  • Consider species-specific reactivity (the HRP-conjugated antibody is human-specific)

• Stress induction protocols:

  • Implement standardized cardiac stress models (hypoxia, oxidative stress, mechanical stretch)

  • Monitor INPP5F expression and localization changes during stress response

  • Correlate with hypertrophic markers and cardiac function parameters

• Multi-parameter analysis:

  • Use INPP5F antibody for protein level quantification via Western blot

  • Combine with cardiomyocyte size measurements and gene expression analysis

  • Monitor downstream pathways (AKT/GSK3B, STAT3) concurrently

• Histological applications:

  • Optimize immunohistochemistry protocols for cardiac tissue sections

  • Consider dual staining with cardiac-specific markers and INPP5F

  • Quantify expression changes across different regions of cardiac tissue

• Functional correlations:

  • Link molecular findings to physiological parameters

  • Correlate INPP5F levels with cardiac function measurements

  • Consider phenotype rescue experiments in INPP5F-modified models

These approaches can help establish INPP5F as a "functionally important modulator of cardiac myocyte size and of the cardiac response to stress" as described in the protein background .

How does INPP5F's regulation of STAT3 signaling impact experimental design when studying inflammatory responses?

When studying INPP5F's negative regulation of STAT3 signaling in inflammatory contexts:

• Experimental timeline considerations:

  • Establish appropriate time points for measuring STAT3 phosphorylation after stimulus

  • Monitor nuclear translocation kinetics of STAT3 in relation to INPP5F activity

  • Design pulse-chase experiments to track signaling dynamics

• Stimulus selection and dosing:

  • Choose appropriate STAT3-activating stimuli (IL-6, IFN-γ, IL-10)

  • Titrate stimulus concentration to avoid overwhelming INPP5F's regulatory capacity

  • Consider combinatorial stimuli that reflect physiological conditions

• Subcellular fractionation approaches:

  • Separate nuclear and cytoplasmic fractions to quantify STAT3 translocation

  • Use the INPP5F antibody to track INPP5F localization during signaling events

  • Correlate phosphoinositide changes with STAT3 nuclear accumulation

• Target gene expression analysis:

  • Design qPCR panels for STAT3-dependent inflammatory genes

  • Compare expression patterns between wild-type and INPP5F-manipulated systems

  • Include time-course analysis to capture primary and secondary response genes

• Phosphorylation site-specific analysis:

  • Use phospho-specific antibodies to distinguish between STAT3 Tyr705 and Ser727 phosphorylation

  • Determine which phosphorylation events are specifically affected by INPP5F

  • Correlate with functionally relevant outcomes

This approach directly addresses INPP5F's role in "negatively regulating STAT3 signaling pathway through inhibition of STAT3 phosphorylation and translocation to the nucleus" as described in the protein background .

How can researchers leverage INPP5F antibodies to investigate its role in stress-induced hypertension?

Recent research has identified lncRNA INPP5F as a key factor inhibiting stress-induced hypertension (SIH) progression. To investigate this connection using antibody-based approaches:

• Expression correlation studies:

  • Quantify INPP5F protein levels in rostral ventrolateral medulla (RVLM) from SIH models

  • Compare with lncRNA INPP5F expression using RT-qPCR

  • Establish relationship between lncRNA expression and protein levels in hypertensive models

• Functional intervention strategies:

  • Design experiments to overexpress lncRNA INPP5F in RVLM

  • Monitor effects on blood pressure, sympathetic nerve activity, and neuronal excitability

  • Use INPP5F antibodies to confirm protein level changes after lncRNA manipulation

• Pathway analysis approach:

  • Investigate the INPP5F/miR-335/Cttn/PI3K-AKT/apoptosis axis

  • Use Western blotting with INPP5F antibody alongside Cttn and phospho-AKT detection

  • Correlate INPP5F protein levels with downstream effectors in the pathway

• Neural apoptosis assessment:

  • Combine INPP5F immunostaining with apoptotic markers

  • Analyze correlation between INPP5F levels and neural survival

  • Establish causative relationships through intervention studies

This approach directly builds on findings that "lncRNA INPP5F was a key factor that inhibited SIH progression, and the identified lncRNA INPP5F/miR-335/Cttn/PI3K-AKT/apoptosis axis represented one of the possible mechanisms" .

What methodological approaches can be used to study INPP5F's function in axon regeneration after CNS injuries?

To investigate INPP5F's potential role as a negative regulator of axon regeneration:

• Expression analysis in injury models:

  • Perform temporal profiling of INPP5F expression after CNS injury

  • Use immunohistochemistry with INPP5F antibody to map expression patterns in injured tissue

  • Compare expression between regeneration-permissive and non-permissive CNS regions

• In vitro neurite outgrowth assays:

  • Establish primary neuronal cultures with INPP5F manipulation (overexpression/knockdown)

  • Quantify neurite length, branching, and growth cone dynamics

  • Challenge neurons with inhibitory substrates mimicking CNS injury environment

• Phosphoinositide dynamics imaging:

  • Utilize phosphoinositide biosensors in combination with INPP5F immunostaining

  • Analyze phosphoinositide distribution in growth cones during regenerative attempts

  • Correlate INPP5F localization with areas of active membrane remodeling

• In vivo intervention studies:

  • Develop targeted INPP5F knockdown strategies for injured CNS tissue

  • Assess axonal regeneration outcomes using tract tracing techniques

  • Combine with functional recovery assessments in animal models

• Downstream pathway analysis:

  • Investigate cytoskeletal regulators potentially affected by INPP5F

  • Focus on interactions with known regeneration-associated genes

  • Consider relationships with other phosphoinositide-modifying enzymes

This experimental framework addresses the hypothesis that INPP5F "may play a role as negative regulator of axon regeneration after central nervous system injuries" .

How might INPP5F antibodies be utilized in developing therapeutic approaches for cardiac stress conditions?

INPP5F antibodies can facilitate therapeutic development for cardiac stress conditions through:

• Therapeutic target validation:

  • Use INPP5F antibodies to confirm target engagement in drug screening assays

  • Monitor INPP5F expression and localization changes during drug treatment

  • Correlate INPP5F modulation with cardioprotective outcomes

• Biomarker development strategy:

  • Evaluate INPP5F as a potential biomarker for cardiac stress using antibody-based detection

  • Develop ELISA protocols using HRP-conjugated antibodies for patient sample analysis

  • Correlate INPP5F levels with disease progression and therapeutic response

• Mechanism-driven therapeutic approaches:

  • Screen compounds that modulate INPP5F-dependent pathways (AKT/GSK3B, STAT3)

  • Use antibody-based assays to monitor pathway activity during compound treatment

  • Validate hits through functional cardiac assays (contractility, hypertrophy)

• Cardiac-specific delivery assessment:

  • Optimize immunohistochemistry protocols to evaluate targeted delivery of INPP5F modulators

  • Quantify cell-type specific effects using co-staining with cardiac markers

  • Monitor off-target effects in non-cardiac tissues

• Translational research considerations:

  • Develop research protocols applicable to human cardiac tissue samples

  • Standardize antibody-based detection for clinical research applications

  • Consider species differences when translating from animal models to humans

This approach builds on INPP5F's established role as a "functionally important modulator of cardiac myocyte size and of the cardiac response to stress" .

What are the most common technical challenges when using HRP-conjugated antibodies in phosphoinositide research?

Research with HRP-conjugated antibodies in phosphoinositide signaling presents several technical challenges:

• Fixation and epitope preservation:

  • Phosphoinositides are sensitive to common fixation methods

  • Recommendation: Use 4% paraformaldehyde with short fixation times (10-15 minutes)

  • Consider specialty fixatives like glutaraldehyde/formaldehyde combinations for phosphoinositide preservation

• Enzymatic activity maintenance:

  • HRP activity can be compromised by storage conditions or contaminants

  • Solution: Store antibody at recommended temperature (typically 4°C with 50% glycerol)

  • Include HRP activity controls in each experiment

• Signal specificity concerns:

  • Phosphoinositide antibodies may show cross-reactivity with structurally similar lipids

  • Approach: Include appropriate negative controls (lipid-depleted samples)

  • Consider peptide competition assays to confirm specificity

• Quantitative analysis limitations:

  • HRP signal can saturate, limiting quantitative range

  • Strategy: Establish standard curves with known concentrations

  • Consider multiple exposure times for Western blots or ELISA readings

• Subcellular localization artifacts:

  • Membranous localization of phosphoinositides creates technical challenges

  • Solution: Use membrane permeabilization protocols optimized for lipid preservation

  • Consider alternative detergents (digitonin, saponin) that preserve membrane structures

These considerations are particularly relevant when studying INPP5F's activities on specific phosphoinositide substrates like phosphatidylinositol 4-phosphate .

How can researchers optimize storage and handling of INPP5F antibody, HRP conjugated to ensure long-term stability and activity?

For optimal storage and handling of INPP5F antibody, HRP conjugated:

• Storage temperature and conditions:

  • Store at recommended temperature (4°C, never freeze)

  • Keep in manufacturer-provided buffer (50% Glycerol, 0.01M PBS, pH 7.4)

  • Protect from light to preserve HRP activity

• Aliquoting strategy:

  • Prepare single-use aliquots to avoid freeze-thaw cycles

  • Use sterile microcentrifuge tubes

  • Include preservative (0.03% Proclin 300 or similar) as in original formulation

• Handling precautions:

  • Avoid contamination with heavy metals, which inhibit HRP

  • Use clean pipette tips and tubes

  • Minimize exposure to strong oxidizing agents

• Activity monitoring:

  • Periodically test activity using simple HRP substrate reactions

  • Compare signal intensity to previous experiments

  • Consider including reference standards in each experiment

• Long-term stability enhancement:

  • Add stabilizing proteins (BSA, 0.1-1%) if diluting stock

  • Consider commercial stabilizers designed for enzyme-conjugated antibodies

  • Document lot-to-lot variation and establish internal controls

Following these practices will help maintain the antibody in optimal condition for experiments involving detection of human INPP5F protein .

What control experiments should be included when using INPP5F antibody in research investigating phosphoinositide signaling networks?

Essential control experiments for INPP5F antibody use in phosphoinositide signaling research:

• Expression manipulation controls:

  • INPP5F knockdown/knockout samples to confirm antibody specificity

  • INPP5F overexpression samples as positive controls

  • Cross-validation with alternative INPP5F antibodies targeting different epitopes

• Substrate specificity controls:

  • In vitro phosphatase assays with defined phosphoinositide substrates

  • Competitive inhibition assays with phosphoinositide analogs

  • Correlation of phosphoinositide levels with INPP5F activity

• Functional pathway controls:

  • Pharmacological inhibitors of AKT/GSK3B pathway to establish pathway specificity

  • OCRL manipulation to examine the functional link between INPP5F and OCRL

  • STAT3 pathway stimulation/inhibition to confirm INPP5F's regulatory role

• Technical controls for HRP-conjugated antibodies:

  • Enzyme activity controls (substrate-only reactions)

  • Non-specific binding controls (isotype-matched irrelevant antibodies)

  • Signal development time-course to establish linearity of detection

• Biological relevance controls:

  • Correlation of INPP5F activity with expected phenotypes (e.g., cardiac stress response)

  • Tissue-specific expression patterns consistent with known biology

  • Developmental or stimulation-dependent expression changes

How can INPP5F antibody be utilized in studying the lncRNA INPP5F/miR-335/Cttn/PI3K-AKT axis in neurological disorders?

To investigate this newly discovered signaling axis in neurological contexts:

• Comprehensive expression profiling:

  • Correlate lncRNA INPP5F, miR-335, and INPP5F protein levels across neurological tissue samples

  • Use RT-qPCR for RNA components and INPP5F antibody for protein detection

  • Create expression maps in various neurological disorder models

• Mechanistic investigation approach:

  • Perform RNA immunoprecipitation to detect lncRNA INPP5F-protein interactions

  • Use INPP5F antibody to identify protein binding partners in neural cells

  • Establish the physical connections between pathway components

• Functional manipulation strategy:

  • Design experiments with selective modulation of pathway components

  • Overexpress/knockdown lncRNA INPP5F while monitoring protein levels

  • Assess miR-335 binding to targets using molecular tools

• Neural cell type-specific analysis:

  • Apply immunofluorescence with INPP5F antibody in brain tissue sections

  • Determine cell type-specific expression patterns

  • Correlate with neurological disorder progression markers

• Therapeutic intervention assessment:

  • Screen compounds that modulate this pathway using antibody-based readouts

  • Evaluate effects on neural apoptosis and excitability

  • Correlate molecular changes with functional outcomes

This approach builds directly on the finding that "lncRNA INPP5F acted as a sponge of miR‐335, which further regulated the Cttn expression" and that this pathway affects "neural apoptosis by activating the PI3K‐AKT pathway" .

What emerging technologies could enhance the application of INPP5F antibodies in spatiotemporal signaling dynamics research?

Cutting-edge technologies for studying spatiotemporal INPP5F dynamics:

• Super-resolution microscopy applications:

  • Implement STORM/PALM techniques with INPP5F antibodies to visualize nanoscale distribution

  • Achieve 10-20nm resolution of INPP5F localization at membrane interfaces

  • Combine with phosphoinositide biosensors for correlated dynamics

• Live-cell imaging approaches:

  • Develop cell-permeable nanobody derivatives of INPP5F antibodies

  • Engineer split-fluorophore systems for detecting protein-protein interactions in real-time

  • Apply FRET/FLIM techniques to monitor INPP5F-substrate proximity

• Mass spectrometry innovations:

  • Implement proximity labeling (BioID, APEX) using INPP5F antibodies

  • Map temporal changes in INPP5F interactome during signaling events

  • Combine with phosphoproteomics to link INPP5F activity to downstream effects

• Microfluidic platform integration:

  • Design microfluidic systems for real-time monitoring of signaling dynamics

  • Apply controlled stimuli while imaging INPP5F localization

  • Perform single-cell analysis of INPP5F-dependent pathways

• Computational modeling enhancement:

  • Develop quantitative models of INPP5F activity in phosphoinositide metabolism

  • Integrate experimental data from antibody-based studies

  • Predict system-level consequences of INPP5F manipulation

These technologies would advance our understanding of INPP5F's dynamic roles in "endocytic recycling" and as a "regulator of TF: TFRC and integrins recycling pathway" involved in "cell migration mechanisms" .

How might multi-omics approaches incorporating INPP5F antibody-based techniques advance our understanding of cardiac and neurological disorders?

Integrative multi-omics strategies using INPP5F antibodies:

• Integrated proteomics and interactomics:

  • Perform immunoprecipitation with INPP5F antibody followed by mass spectrometry

  • Map INPP5F protein interaction networks in health and disease

  • Compare interactomes across cardiac and neurological tissues

• Spatial transcriptomics correlation:

  • Combine INPP5F immunohistochemistry with spatial transcriptomics

  • Map regional variations in INPP5F protein levels and gene expression profiles

  • Identify tissue microenvironments with altered INPP5F signaling

• Epigenomic integration:

  • Correlate INPP5F protein levels with epigenetic modifications at the INPP5F locus

  • Investigate regulatory mechanisms controlling INPP5F expression

  • Identify disease-associated epigenetic signatures

• Single-cell multi-parameter analysis:

  • Develop protocols for simultaneous detection of INPP5F protein, lncRNA INPP5F, and miR-335

  • Apply to patient-derived samples and disease models

  • Identify cell populations with altered signaling profiles

• Clinical correlative studies:

  • Establish INPP5F protein quantification protocols using HRP-conjugated antibodies

  • Correlate with clinical parameters in cardiac and neurological patients

  • Develop prognostic models incorporating molecular and clinical data

This integrated approach would advance understanding of how INPP5F functions as both a "modulator of cardiac myocyte size and of the cardiac response to stress" and potentially as a "negative regulator of axon regeneration after central nervous system injuries" .