INPP5K Antibody

What is INPP5K and why is it important in cellular research?

INPP5K (Inositol Polyphosphate-5-Phosphatase K) is an endoplasmic reticulum (ER)-residing phosphoinositide 5-phosphatase that dephosphorylates PI(4,5)P2, thereby regulating ER morphology. This enzyme plays a crucial role in connecting the endoplasmic reticulum to microtubules through its interaction with β-tubulin via its 5-phosphatase domain. The significance of INPP5K in research stems from its involvement in ER remodeling processes, which impacts neuronal development and may be implicated in hereditary spastic paraplegia, an inherited neuronal disorder . Understanding INPP5K function provides insights into fundamental cellular processes related to ER morphology, microtubule interactions, and potentially, disease mechanisms.

What types of INPP5K antibodies are available for research applications?

Several variants of INPP5K antibodies are available for research, differing in host species, clonality, binding regions, and applications. The primary options include:

Researchers should select antibodies based on specific experimental requirements, including the desired epitope region and application methodology .

How do I determine which INPP5K antibody specificity is best for my research?

Selecting the appropriate INPP5K antibody specificity depends on several experimental considerations:

• Target domain interest: If investigating specific INPP5K domains (e.g., 5-phosphatase domain or SKICH domain), choose antibodies that recognize epitopes within these regions. For 5-phosphatase domain studies, antibodies targeting AA 1-317 region would be preferable, while SKICH domain studies might benefit from antibodies targeting AA 318-448 .

• Cross-reactivity requirements: Consider whether cross-reactivity with INPP5K from multiple species is necessary. Some antibodies are human-specific, while others react with mouse, rat, and other species .

• Application compatibility: Ensure the antibody is validated for your specific application (WB, IF, IHC, ELISA). For instance, if performing co-localization studies of INPP5K with microtubules, an antibody validated for immunofluorescence would be essential .

• Epitope accessibility: For studies examining protein-protein interactions, consider whether your antibody's epitope might be masked by interaction partners. In studies of INPP5K-tubulin interactions, for example, antibodies targeting regions away from the interaction interface (AA 261-264) might yield more consistent results .

• Mutation studies: If your research involves INPP5K mutants (such as D310G or 4A mutants), ensure your antibody's epitope is not affected by these mutations to avoid false negative results .

What are the validated applications for INPP5K antibodies in research?

INPP5K antibodies have been validated for several key applications in molecular and cellular biology research:

• Western Blotting (WB): Most INPP5K antibodies are validated for western blot applications, allowing detection of native and recombinant INPP5K proteins. This application is useful for confirming knockdown efficiency, overexpression validation, and protein level quantification .

• Immunofluorescence (IF): Several INPP5K antibodies are validated for immunofluorescence microscopy, enabling visualization of subcellular localization. This is particularly valuable for co-localization studies with microtubules or ER markers, as demonstrated in research showing INPP5K co-localization with α-tubulin, especially at the cell periphery .

• Immunohistochemistry (IHC): Some antibodies are suitable for tissue section analysis, allowing investigation of INPP5K expression patterns in different tissues and cell types .

• Immunoprecipitation (IP): INPP5K antibodies can be used for pull-down assays to study protein-protein interactions, as evidenced by studies identifying β-tubulin as an INPP5K-binding protein through immunoprecipitation followed by mass spectrometry .

• ELISA: Several antibodies are validated for enzyme-linked immunosorbent assays, enabling quantitative measurement of INPP5K protein levels .

Each application requires specific optimization steps and may benefit from particular antibody characteristics (monoclonal vs. polyclonal, specific epitope targeting).

How can I optimize Western blot protocols for INPP5K detection?

Optimizing Western blot protocols for INPP5K detection requires attention to several key factors:

• Sample preparation:

  • Include phosphatase inhibitors in lysis buffers to preserve the native state of this phosphatase

  • For membrane-associated INPP5K, use detergent-containing buffers (e.g., RIPA with 0.1-0.5% SDS)

  • Heat samples at 95°C for 5 minutes in reducing buffer to ensure complete denaturation

• Gel selection and transfer:

  • Use 10-12% polyacrylamide gels for optimal resolution of INPP5K (molecular weight ~50 kDa)

  • For transfer to PVDF membranes, a semi-dry transfer system at 15V for 30-45 minutes typically yields good results

• Antibody dilution and incubation:

  • Primary antibody dilutions typically range from 1:500 to 1:2000

  • Overnight incubation at 4°C generally produces cleaner results than short incubations

  • Use 5% BSA in TBST rather than milk for blocking and antibody dilution to reduce background

• Detection considerations:

  • When analyzing INPP5K mutants (e.g., 4A mutant), ensure the antibody's epitope isn't affected by the mutation

  • For co-immunoprecipitation experiments, use antibodies targeting different epitopes for immunoprecipitation and detection

  • When studying INPP5K-tubulin interactions, consider potential interference from binding partners

• Controls:

  • Include positive controls (cells known to express INPP5K)

  • Include negative controls (INPP5K knockdown cells)

  • For overexpression studies, include both tagged and untagged versions to account for tag interference

These optimizations help ensure specific, reproducible detection of INPP5K in Western blot applications.

What is the best approach for immunofluorescence experiments using INPP5K antibodies?

For optimal immunofluorescence results with INPP5K antibodies, follow these methodological recommendations:

• Cell preparation and fixation:

  • Fix cells with 3.7% formaldehyde for 10-15 minutes at room temperature

  • Permeabilize with PBS containing 0.2% Triton X-100 for 10 minutes

  • Block with 1% FBS in PBS for 30 minutes to reduce non-specific binding

• Antibody selection and incubation:

  • Choose INPP5K antibodies specifically validated for immunofluorescence

  • Incubate with primary antibody for 1 hour at room temperature or overnight at 4°C

  • Use appropriate fluorophore-conjugated secondary antibodies matching the host species of your primary antibody

  • For co-localization studies with cytoskeletal elements, consider dual staining with anti-α-tubulin antibodies

• Co-localization studies:

  • When studying INPP5K-microtubule co-localization, counterstain with anti-α-tubulin antibodies

  • For ER studies, co-stain with ER markers such as DsRed2-ER (containing ER-targeting sequence of calreticulin and KDEL retention signal)

  • For actin co-localization, use fluorophore-conjugated phalloidin (e.g., AlexaFluor-647-labeled phalloidin)

• Imaging and analysis:

  • Use confocal microscopy for optimal resolution of subcellular structures

  • Consider using a 60× oil immersion objective with NA 1.35 for high-resolution imaging

  • For quantitative analysis of ER morphology, measure the area occupied by ER markers using software like ImageJ

  • Analyze at least 30 cells per condition for statistical validity

• Controls and validation:

  • Include INPP5K knockdown cells as negative controls

  • Use overexpression of wild-type and mutant INPP5K constructs (e.g., INPP5K 4A) to validate antibody specificity

  • Consider comparing results from multiple INPP5K antibodies targeting different epitopes

Following these guidelines will help ensure high-quality, reproducible immunofluorescence imaging of INPP5K and its interactions with cellular structures.

What are the key functional domains of INPP5K and how do they affect antibody selection?

INPP5K contains two primary functional domains that should inform antibody selection strategies:

• N-terminal 5-phosphatase catalytic domain (amino acids 1-317):

  • Responsible for the enzymatic activity that dephosphorylates PI(4,5)P2

  • Contains a basic amino acid cluster (K261/K262/R263/K264) critical for tubulin binding

  • Antibodies targeting this domain are useful for studying INPP5K enzymatic function and tubulin interactions

  • For mutation studies involving the phosphatase-dead D310G variant, antibodies targeting regions away from this mutation site should be selected

• C-terminal SKICH domain (amino acids 318-448):

  • Mediates localization to the ER and plasma membrane

  • Interacts with the ER membrane protein Arl6IP1

  • Antibodies recognizing this domain are valuable for studies focusing on INPP5K subcellular localization

  • The D361A mutation affects ER localization but not tubulin binding

When selecting antibodies, researchers should consider:

• For studying enzymatic activity: Choose antibodies targeting regions outside the active site to avoid interference with substrate binding

• For localization studies: Select antibodies that recognize the SKICH domain or full-length protein

• For tubulin interaction studies: Avoid antibodies targeting the basic amino acid cluster (K261-K264) as this region mediates tubulin binding and may be masked in complexes

• For studies involving specific mutants: Ensure the antibody's epitope is not affected by the mutation of interest

Understanding these domain-specific considerations ensures more effective antibody selection and experimental design.

How does INPP5K interact with microtubules and what implications does this have for experimental design?

INPP5K interacts with microtubules through a specific mechanism that has important experimental implications:

• Interaction mechanism:

  • INPP5K binds directly to β-tubulin through its 5-phosphatase domain

  • The interaction is mediated by a basic amino acid cluster (K261/K262/R263/K264) within the phosphatase domain

  • Mutation of these residues (INPP5K 4A mutant) abolishes interaction with both α- and β-tubulin

  • This interaction is independent of INPP5K's ER localization function, as the ER-delocalizing INPP5K D361A mutant still immunoprecipitates with tubulin

• Experimental implications:

  • When designing co-immunoprecipitation experiments, consider that antibodies targeting the K261-K264 region may disrupt or fail to detect the INPP5K-tubulin interaction

  • For immunofluorescence studies, EGFP-tagged INPP5K shows co-localization with α-tubulin, particularly at the cell periphery

  • The phosphatase domain alone (INPP5K 1-317) localizes to microtubules, while the SKICH domain alone (INPP5K 318-448) does not

  • Both domains appear necessary for proper INPP5K anchoring at the ER membrane

• Functional significance:

  • INPP5K likely serves as a connector between microtubules and the ER

  • INPP5K knockdown results in ER remodeling: decreased ER tubules and increased ER sheets

  • Re-expression of wild-type INPP5K, but not the tubulin-binding defective 4A mutant, rescues normal ER morphology

• Technical considerations:

  • For live-cell imaging of INPP5K-microtubule interactions, fusion proteins should be designed to avoid disrupting the K261-K264 region

  • When studying ER morphology, quantification should include measurement of the percentage area occupied by the ER in the cytoplasm using markers like DsRed2-ER

  • Statistical analysis should compare at least 30 cells per condition to account for cell-to-cell variability

Understanding these interaction details ensures proper experimental design when studying INPP5K's role in connecting the ER to microtubules.

What is the relationship between INPP5K's phosphatase activity and its role in ER morphology regulation?

The relationship between INPP5K's phosphatase activity and its regulation of ER morphology involves several interconnected mechanisms:

This multifaceted relationship between INPP5K's enzymatic activity and structural role makes it a fascinating but complex subject for research into ER biology.

How can INPP5K antibodies be used to study ER-microtubule interactions in disease models?

INPP5K antibodies can be leveraged in sophisticated ways to investigate ER-microtubule interactions in disease models, particularly for neurological disorders:

• Neurological disease applications:

  • Given INPP5K's role in ER remodeling and potential implications for hereditary spastic paraplegia , antibodies can be used to examine altered INPP5K localization or function in disease models

  • In neuronal cells, INPP5K antibodies can help visualize ER distribution in axons and dendrites, where ER remodeling is critical for normal development and function

  • Comparison of INPP5K localization patterns between healthy and disease model neurons may reveal pathogenic mechanisms

• Advanced imaging approaches:

  • Super-resolution microscopy (STORM, PALM) using INPP5K antibodies can resolve nanoscale details of ER-microtubule contact sites

  • Multi-color imaging combining INPP5K antibodies with ER markers (e.g., DsRed2-ER) and microtubule markers can reveal triple co-localization patterns

  • Live-cell imaging in disease models can capture dynamic changes in INPP5K-mediated ER-microtubule interactions

• Biochemical interaction studies:

  • Proximity ligation assays (PLA) using INPP5K antibodies paired with anti-tubulin antibodies can quantitatively assess INPP5K-microtubule interactions in situ

  • Co-immunoprecipitation with INPP5K antibodies followed by mass spectrometry can identify altered interaction partners in disease states

  • Comparative interaction mapping between normal and disease-associated INPP5K variants can reveal mechanistic insights

• Methodological considerations:

  • For studies in primary neurons or disease models, careful antibody validation is essential

  • When comparing diseased versus healthy tissues, standardized immunostaining protocols must be maintained

  • Quantitative analysis should include multiple parameters beyond co-localization, such as ER tubule length, branch points, and sheet-to-tubule ratios

  • Controls should include INPP5K knockdown and rescue with wild-type or mutant constructs

• Therapeutic implications:

  • INPP5K antibodies can help evaluate whether therapeutic candidates restore normal INPP5K localization and ER-microtubule interactions

  • Time-course studies can determine whether therapeutic interventions halt or reverse pathological ER remodeling

These advanced applications of INPP5K antibodies can provide crucial insights into disease mechanisms involving ER-microtubule interactions and potential therapeutic approaches.

What strategies can resolve conflicting results when using different INPP5K antibodies?

Researchers may encounter conflicting results when using different INPP5K antibodies. The following systematic approach can help resolve such discrepancies:

• Epitope mapping and antibody characterization:

  • Determine the exact epitopes recognized by each antibody

  • Epitopes within functional domains (e.g., the K261-K264 basic cluster) may be masked by protein-protein interactions

  • Different antibodies may preferentially recognize specific conformational states of INPP5K

  • Some epitopes may be affected by post-translational modifications

• Validation strategy:

  • Perform parallel experiments with multiple antibodies targeting different INPP5K regions

  • Include INPP5K knockout/knockdown controls to confirm specificity

  • Test antibodies against overexpressed wild-type and mutant INPP5K constructs

  • Consider peptide competition assays to confirm epitope specificity

• Technical optimization:

  • Systematically compare fixation methods for immunofluorescence (formaldehyde, methanol, glutaraldehyde)

  • Test different detergents and concentrations for membrane permeabilization

  • Optimize blocking conditions to minimize background signal

  • For western blotting, compare reducing versus non-reducing conditions

• Reconciliation approaches:

  • When antibodies yield different subcellular localization patterns, consider that each may be detecting distinct INPP5K subpopulations

  • For quantitative discrepancies, normalize results using internal controls

  • When antibodies disagree on protein-protein interactions, consider that the antibody itself may disrupt interactions

  • For conflicting results in disease models, consider tissue-specific or context-dependent factors

• Integration with complementary methods:

  • Corroborate antibody-based findings with tagged INPP5K constructs

  • Validate protein-protein interactions using reciprocal co-immunoprecipitation

  • Confirm subcellular localization using cell fractionation followed by western blotting

  • Implement CRISPR-Cas9 tagging of endogenous INPP5K for definitive localization studies

This systematic approach helps distinguish between true biological phenomena and technical artifacts when different INPP5K antibodies yield conflicting results.

How can quantitative measurements be performed to assess INPP5K-mediated ER morphology changes?

Quantitative assessment of INPP5K-mediated ER morphology changes requires rigorous methodological approaches:

• Image acquisition parameters:

  • Use confocal microscopy with consistent settings across samples

  • A 60× oil immersion objective with NA 1.35 provides suitable resolution for ER structures

  • Z-stack acquisition (0.2-0.3 μm steps) ensures capture of the full ER network

  • Include appropriate controls (INPP5K knockdown, wild-type rescue, mutant rescue) in each experiment

• ER visualization techniques:

  • Transfect cells with fluorescent ER markers like DsRed2-ER (containing calreticulin ER-targeting sequence and KDEL retention signal)

  • For endogenous ER labeling, use antibodies against resident ER proteins (calnexin, calreticulin, Sec61β)

  • For live-cell imaging, consider ER-Tracker dyes or genetically encoded ER markers

• Quantitative metrics:

  • ER area: Measure the percentage of cytoplasmic area occupied by ER markers using software like ImageJ (minimum 30 cells per condition)

  • ER morphology classification: Calculate the ratio of peripheral ER tubules to perinuclear ER sheets

  • Network analysis: Quantify ER tubule length, number of branch points, and polygonal network characteristics

  • 3D reconstruction: Calculate ER volume and surface area from z-stack images

• Statistical analysis:

  • Compare treatments using appropriate statistical tests (t-test for two conditions, ANOVA for multiple conditions)

  • Present data as mean ± standard deviation with clear indication of sample sizes

  • Consider cell-to-cell variability by analyzing sufficient numbers of cells (>30 per condition)

  • Account for variations in cell size and shape through normalization

• Advanced analytical approaches:

  • Time-lapse imaging to capture dynamic ER remodeling in response to INPP5K manipulation

  • Fluorescence recovery after photobleaching (FRAP) to assess ER membrane dynamics

  • Correlative light and electron microscopy (CLEM) for ultrastructural validation of fluorescence-based findings

  • Machine learning algorithms for unbiased classification of ER morphological patterns

• Experimental design for conclusive results:

  • Compare INPP5K knockdown cells with control cells

  • Include rescue experiments with wild-type INPP5K, phosphatase-dead D310G mutant, and tubulin-binding defective 4A mutant

  • Assess effects under both basal and stimulated conditions (e.g., insulin stimulation)

  • Consider cell type-specific effects by comparing multiple cell lines

These quantitative approaches enable robust assessment of how INPP5K influences ER morphology, providing insights into both normal cellular physiology and potential disease mechanisms.

How can INPP5K antibodies be applied in neurodevelopmental research?

INPP5K antibodies offer valuable tools for investigating neurodevelopmental processes, particularly given INPP5K's potential role in neurological disorders:

• Neuronal differentiation studies:

  • Track INPP5K expression and localization during different stages of neuronal differentiation

  • Examine co-localization with microtubules in developing axons and dendrites, where ER distribution is critical

  • Compare INPP5K distribution in different neuronal subtypes to identify cell type-specific functions

  • Correlate INPP5K localization patterns with key developmental transitions

• Neurite development applications:

  • Given that ER remodeling is important for axon and dendrite development , INPP5K antibodies can help visualize ER distribution during neurite outgrowth

  • Use INPP5K antibodies in combination with cytoskeletal markers to study the coordination between ER extension and the developing cytoskeleton

  • Time-course studies can reveal how INPP5K-mediated ER-microtubule interactions change during neurite elongation and branching

• Methodological approaches:

  • Primary neuron cultures provide an ideal system for studying INPP5K during authentic neurodevelopmental processes

  • Brain slice cultures maintain tissue architecture while allowing antibody access for immunostaining

  • For in vivo studies, consider immunohistochemistry on brain sections at different developmental stages

  • Combine INPP5K immunostaining with markers for specific developmental stages or neuronal subtypes

• Disease model applications:

  • Compare INPP5K localization in neurons derived from healthy individuals versus those with neurodevelopmental disorders

  • Patient-derived induced pluripotent stem cells (iPSCs) differentiated into neurons offer a valuable model system

  • Examine whether disease-associated INPP5K variants show altered localization or ER morphology phenotypes

  • Consider INPP5K's potential role in hereditary spastic paraplegia when designing studies

• Advanced imaging techniques:

  • Super-resolution microscopy can resolve ER-microtubule contact sites in fine neuronal processes

  • Live-cell imaging in developing neurons can capture dynamic INPP5K-dependent ER remodeling events

  • 3D reconstruction from confocal z-stacks can provide comprehensive views of INPP5K distribution throughout complex neuronal architectures

These applications can help elucidate INPP5K's role in neurodevelopment and potentially identify mechanisms underlying neurodevelopmental disorders.

What are the critical controls and validations when studying INPP5K mutants with antibodies?

When studying INPP5K mutants with antibodies, rigorous controls and validations are essential for reliable interpretation:

• Antibody epitope considerations:

  • Verify that the mutation does not alter the antibody's epitope, which could lead to false negative results

  • For mutations in or near the antibody binding site, use multiple antibodies targeting different regions

  • Consider using tagged constructs (FLAG, GFP) as alternative detection methods

  • Perform western blots to confirm that antibodies detect mutant proteins with similar efficiency as wild-type

• Expression level validations:

  • Quantify expression levels of wild-type and mutant INPP5K to ensure comparable expression

  • For transient transfections, normalize to a co-transfected marker or use FACS sorting to select cells with similar expression levels

  • In stable cell lines, confirm similar expression levels by qPCR and western blotting

  • Consider using inducible expression systems to achieve controlled expression levels

• Functional control experiments:

  • For phosphatase-dead mutants (e.g., D310G), confirm loss of enzymatic activity using phosphatase assays

  • For tubulin-binding mutants (e.g., 4A mutant), validate loss of tubulin interaction by co-immunoprecipitation

  • For localization mutants (e.g., D361A), confirm altered subcellular distribution by co-staining with appropriate markers

  • Include rescue experiments in INPP5K knockdown backgrounds to assess functional complementation

• Specificity controls:

  • Use INPP5K knockout/knockdown cells as negative controls

  • Include competitive blocking with immunizing peptides when available

  • Perform parallel experiments with non-specific IgG from the same species

  • For novel mutants, include well-characterized mutants (D310G, 4A) as reference points

• Technical considerations:

  • For immunofluorescence studies of mutants, standardize image acquisition parameters across samples

  • When comparing wild-type and mutant localization, acquire images in the same experiment under identical conditions

  • For co-localization studies, include appropriate controls for bleed-through between channels

  • When quantifying phenotypes, blind the analysis to prevent bias

These controls and validations ensure that observed differences between wild-type and mutant INPP5K reflect genuine biological phenomena rather than technical artifacts, leading to more robust and reproducible findings.

How can researchers incorporate INPP5K antibodies in studies of stress-induced ER remodeling?

INPP5K antibodies can be strategically incorporated into studies of stress-induced ER remodeling, providing insights into adaptive and pathological responses:

• Stress induction protocols:

  • Examine INPP5K localization and function during canonical ER stressors (tunicamycin, thapsigargin, DTT)

  • Assess INPP5K's role during physiological stresses (glucose deprivation, hypoxia, oxidative stress)

  • Investigate whether INPP5K undergoes relocation or modification during the unfolded protein response (UPR)

  • Compare acute versus chronic stress effects on INPP5K-mediated ER-microtubule interactions

• Co-visualization strategies:

  • Combine INPP5K antibodies with markers of ER stress (phospho-PERK, phospho-IRE1α, ATF6 cleavage)

  • Co-stain for ER chaperones (GRP78/BiP, which has been identified as an INPP5K-interacting protein)

  • Visualize INPP5K alongside markers of ER morphology (DsRed2-ER) during stress progression

  • Track INPP5K localization relative to microtubules during stress-induced ER remodeling

• Functional investigation approaches:

  • Compare stress-induced ER remodeling in control versus INPP5K knockdown cells

  • Assess whether INPP5K phosphatase activity changes during ER stress

  • Determine if stress alters INPP5K's interaction with tubulin or other binding partners

  • Investigate whether phosphatase-dead (D310G) or tubulin-binding defective (4A) mutants show altered stress responses

• Temporal analysis methods:

  • Implement time-course studies to track INPP5K localization during stress induction, peak response, and recovery

  • Use live-cell imaging with transiently expressed fluorescent INPP5K constructs alongside fixed-cell antibody validation

  • Assess whether INPP5K plays different roles in early versus late phases of the stress response

  • Determine if INPP5K is involved in the restoration of normal ER morphology during stress recovery

• Quantitative assessment techniques:

  • Measure changes in INPP5K-tubulin co-localization coefficients during stress

  • Quantify ER morphology parameters (tubular/sheet ratio, network complexity) in relation to INPP5K status

  • Assess cell viability and stress marker expression in relation to INPP5K function

  • Compare stress adaptation in cells expressing wild-type versus mutant INPP5K

By incorporating these approaches, researchers can elucidate INPP5K's role in linking stress sensing to ER remodeling, potentially revealing therapeutic targets for diseases involving ER stress.

What are the latest research technologies that can be combined with INPP5K antibodies?

Cutting-edge technologies can be integrated with INPP5K antibodies to advance understanding of its function and regulation:

• Proximity labeling techniques:

  • BioID or TurboID fusions to INPP5K can identify proteins in its proximity at ER-microtubule contact sites

  • APEX2-INPP5K fusion followed by electron microscopy can visualize INPP5K's ultrastructural context

  • Split-BioID approaches can identify proteins at specific INPP5K interaction interfaces (e.g., INPP5K-tubulin interface)

  • Validation of proximity labeling results can be performed using conventional co-immunoprecipitation with INPP5K antibodies

• Advanced microscopy methods:

  • Lattice light-sheet microscopy can capture INPP5K dynamics with minimal phototoxicity in living cells

  • Expansion microscopy can physically enlarge specimens to visualize INPP5K's nanoscale organization

  • Cryo-electron tomography combined with INPP5K immunogold labeling can reveal its native structural context

  • Fluorescence correlation spectroscopy can measure INPP5K diffusion dynamics and complex formation

• Genome editing applications:

  • CRISPR-Cas9 knock-in of epitope tags or fluorescent proteins at the endogenous INPP5K locus

  • Base editors or prime editors for introducing specific point mutations (e.g., 4A or D310G) in endogenous INPP5K

  • CRISPR interference or activation to modulate endogenous INPP5K expression

  • Validation of edited cells using INPP5K antibodies to confirm desired modifications

• Single-cell analysis techniques:

  • Imaging mass cytometry with INPP5K antibodies can profile its expression across heterogeneous cell populations

  • Single-cell RNA-seq paired with INPP5K protein analysis can correlate transcriptional states with protein levels

  • Spatial transcriptomics combined with INPP5K immunofluorescence can map expression patterns within tissues

  • Mass spectrometry imaging can visualize INPP5K distribution in tissue sections with high molecular specificity

• Functional screening approaches:

  • CRISPR screens targeting INPP5K interactors followed by phenotypic analysis

  • Small molecule screens for compounds that modulate INPP5K localization or function

  • Synthetic genetic interaction mapping to identify genes that buffer or enhance INPP5K-related phenotypes

  • Validation of hits from these screens using INPP5K antibodies for biochemical and imaging assays

These emerging technologies, when combined with well-characterized INPP5K antibodies, offer unprecedented opportunities to dissect INPP5K's functions and regulatory mechanisms with molecular precision.

How can phosphoproteomic analysis be integrated with INPP5K antibody studies?

Integrating phosphoproteomic analysis with INPP5K antibody studies provides powerful insights into its signaling context and functional impacts:

• Phosphorylation status of INPP5K:

  • Immunoprecipitate INPP5K using validated antibodies followed by mass spectrometry to identify phosphorylation sites

  • Compare phosphorylation patterns under different cellular conditions (serum starvation, insulin stimulation)

  • Generate phospho-specific antibodies against key INPP5K phosphorylation sites

  • Investigate whether phosphorylation affects INPP5K's enzymatic activity, localization, or protein interactions

• Phosphoproteome changes upon INPP5K manipulation:

  • Compare global phosphoproteomes between control and INPP5K knockdown cells

  • Analyze phosphoproteome changes when overexpressing wild-type versus mutant INPP5K (D310G, 4A)

  • Focus on phosphorylation changes in proteins associated with ER morphology and microtubule dynamics

  • Validate key phosphorylation changes using phospho-specific antibodies

• Substrate identification and validation:

  • Identify potential direct and indirect targets of INPP5K phosphatase activity

  • Compare phosphoinositide profiles between control and INPP5K-manipulated cells

  • Investigate whether INPP5K affects the phosphorylation status of its binding partners (e.g., tubulin)

  • Use in vitro phosphatase assays with immunoprecipitated INPP5K to confirm direct substrates

• Temporal dynamics analysis:

  • Perform time-course phosphoproteomic analysis following INPP5K activation or inhibition

  • Correlate phosphorylation changes with alterations in ER morphology

  • Identify rapid versus delayed phosphorylation events to distinguish direct and indirect effects

  • Use INPP5K antibodies to track its localization during signaling dynamics

• Integrated multi-omics approaches:

  • Combine phosphoproteomics with interactomics data from INPP5K immunoprecipitation studies

  • Integrate transcriptomic responses to INPP5K manipulation with phosphoproteomic changes

  • Correlate changes in protein abundance (proteomics) with phosphorylation status

  • Use network analysis to identify signaling hubs affected by INPP5K activity

This integration of phosphoproteomics with INPP5K antibody studies provides a systems-level understanding of how INPP5K functions within cellular signaling networks and affects downstream processes like ER morphology regulation.

What are the unsolved questions regarding INPP5K function that future antibody-based studies might address?

Several critical questions about INPP5K remain unanswered and represent promising avenues for future antibody-based investigations:

• Regulatory mechanisms of INPP5K:

  • How is INPP5K activity regulated in response to cellular signals?

  • Are there tissue-specific mechanisms controlling INPP5K localization or function?

  • Does INPP5K undergo conformational changes that affect its dual roles in phosphatase activity and microtubule binding?

  • What post-translational modifications influence INPP5K and how do they affect its functions?

• Disease-relevant mechanisms:

  • How do disease-associated INPP5K mutations affect its localization, interactions, and function?

  • Does INPP5K play different roles in different cell types relevant to hereditary spastic paraplegia?

  • Are there compensation mechanisms when INPP5K function is compromised?

  • How does INPP5K contribute to ER stress responses in disease contexts?

• Dynamic behavior during cellular processes:

  • How does INPP5K localization and function change during cell division?

  • What role does INPP5K play during cell migration, when ER and microtubule dynamics are crucial?

  • How is INPP5K involved in specialized cellular processes like neuronal synaptic plasticity?

  • Does INPP5K function change during cellular differentiation or development?

• Interaction network complexity:

  • Beyond tubulin and GRP78/BiP, what other proteins interact with INPP5K?

  • Do INPP5K's interaction partners differ between cell types or cellular compartments?

  • How do protein interactions regulate INPP5K's dual functions?

  • Are there competitive interactions that determine INPP5K's localization or activity?

• Therapeutic targeting potential:

  • Can modulation of INPP5K activity or localization provide therapeutic benefits in disease models?

  • Are there INPP5K-dependent processes that could be targeted pharmacologically?

  • How does INPP5K respond to existing drugs that affect ER or microtubule function?

  • Could INPP5K serve as a biomarker for disease progression or therapeutic response?

Future antibody-based studies, particularly those employing new technologies and methodologies, have the potential to address these questions and advance our understanding of INPP5K's fundamental biology and disease relevance.

What are the emerging trends in INPP5K research that researchers should be aware of?

Researchers working with INPP5K antibodies should be aware of several emerging trends that are shaping the field:

• Expanded disease associations:

  • Beyond hereditary spastic paraplegia, INPP5K is being investigated in other neurological disorders

  • Connections to insulin signaling and metabolism suggest potential roles in metabolic diseases

  • The link between ER stress and various diseases positions INPP5K as a potential player in conditions from neurodegeneration to cancer

  • Researchers should monitor new disease associations to identify relevant model systems

• Integration with membrane contact site biology:

  • Growing recognition of INPP5K as a component of ER-microtubule contact sites

  • Potential roles at other membrane contact sites (ER-plasma membrane, ER-mitochondria)

  • Investigation of INPP5K's contribution to interorganelle communication

  • Collaboration with researchers studying other contact site proteins may yield synergistic insights

• Systems biology approaches:

  • Shift from studying INPP5K in isolation to understanding its position in cellular networks

  • Integration of phosphoinositide signaling with other signaling pathways

  • Computational modeling of how INPP5K activity affects membrane dynamics

  • Multi-omics approaches to comprehensively map INPP5K's cellular impacts

• Technological innovations:

  • Development of sensors to monitor INPP5K activity in living cells

  • Application of cryo-electron microscopy to resolve INPP5K structure and interactions

  • Implementation of optogenetic tools to acutely manipulate INPP5K function

  • Improved phosphoinositide imaging techniques to visualize INPP5K substrates

• Therapeutic targeting developments:

  • Screening for small molecules that modulate INPP5K activity or interactions

  • Exploration of gene therapy approaches for INPP5K-related disorders

  • Investigation of whether existing drugs affecting ER or microtubule function modulate INPP5K

  • Development of peptide inhibitors targeting specific INPP5K interaction interfaces

Staying informed about these emerging trends will help researchers design forward-looking studies that contribute meaningfully to the rapidly evolving INPP5K field.

What are the recommended best practices for publishing research involving INPP5K antibodies?

To ensure reliability and reproducibility in research using INPP5K antibodies, researchers should adhere to these best practices for publication:

• Comprehensive antibody reporting:

  • Provide complete antibody information including vendor, catalog number, lot number, and RRID (Research Resource Identifier)

  • Specify the immunogen used to generate the antibody and the species of origin

  • Indicate whether the antibody is monoclonal or polyclonal

  • Describe any modifications (e.g., conjugation to fluorophores or enzymes)

• Validation documentation:

  • Include explicit validation data demonstrating antibody specificity

  • Show controls using INPP5K knockdown/knockout samples

  • For novel applications, validate using overexpressed wild-type and mutant INPP5K

  • When multiple antibodies are available, demonstrate consistent results or explain discrepancies

• Detailed methodological reporting:

  • Provide complete protocols including antibody dilutions, incubation times and temperatures

  • For western blotting, specify blocking conditions, washing protocols, and detection methods

  • For immunofluorescence, detail fixation, permeabilization, and mounting procedures

  • For immunoprecipitation, describe lysis conditions, bead type, and washing stringency

• Imaging and quantification transparency:

  • Include representative images showing the full dynamic range of signals

  • Provide clear descriptions of image acquisition parameters (exposure times, gain settings)

  • Detail any image processing steps applied post-acquisition

  • For quantitative analyses, explain measurement methods and statistical approaches in detail

• Results interpretation guidelines:

  • Acknowledge limitations of antibody-based approaches

  • Consider alternative explanations for unusual or unexpected findings

  • Discuss how antibody characteristics might influence interpretation of results

  • Place findings in context of previous work, addressing any apparent contradictions

• Data sharing practices:

  • Consider depositing original, unprocessed image files in appropriate repositories

  • Share detailed protocols on platforms like protocols.io

  • Make custom antibodies available to other researchers when possible

  • Provide raw data underlying quantitative analyses as supplementary material