INPP5B Antibody

What is INPP5B and why is it important for cellular function?

INPP5B (Type II inositol polyphosphate 5-phosphatase) preferentially hydrolyzes the 5-phosphate of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3], as well as soluble inositol phosphates . INPP5B plays crucial roles in multiple cellular processes including membrane trafficking in the early secretory pathway, actin cytoskeleton remodeling, and B cell receptor signaling .

The significance of INPP5B is highlighted by its close homology to OCRL1, the protein mutated in the X-linked disorder oculocerebrorenal syndrome of Lowe. INPP5B and OCRL1 share 45% sequence identity, similar domain architecture, and overlapping substrate specificity . While INPP5B knockout mice show relatively mild phenotypes (male sterility in certain genetic backgrounds), the combined knockout of both INPP5B and OCRL1 results in embryonic lethality, indicating these enzymes can functionally compensate for each other in vivo .

What cellular compartments should researchers target when using INPP5B antibodies?

When designing experiments with INPP5B antibodies, researchers should consider its complex subcellular distribution. INPP5B localizes to:

• The Golgi apparatus, with enrichment on the cis-side (showing better colocalization with GM130 than TGN46)

• The ER-to-Golgi intermediate compartment (ERGIC)

• Plasma membrane ruffles induced by growth factors

• Enlarged endosomes seen upon expression of constitutively active RAB5

• A significant fraction appears cytosolic

This distribution pattern differs from its homolog OCRL1, which shows greater enrichment at the trans-Golgi network. When performing immunolocalization studies, researchers should include appropriate markers for these compartments (GM130 for cis-Golgi, TGN46 for trans-Golgi, ERGIC53 for ERGIC) to precisely identify INPP5B localization .

How can researchers validate INPP5B antibody specificity?

Thorough validation of INPP5B antibodies is essential to ensure reliable experimental results. Methods should include:

• Western blotting to confirm detection of a single band at the expected molecular weight (~84 kDa)

• Comparison with tagged recombinant INPP5B as a positive control

• Testing in INPP5B-depleted cells (knockout or knockdown) as negative controls

• Assessing cross-reactivity with the closely related OCRL1 protein

• Using multiple antibodies targeting different epitopes when possible

• Testing fixation-dependent epitope accessibility (both methanol and paraformaldehyde fixation methods have been successfully used for INPP5B immunostaining)

What protein domains should researchers target when selecting INPP5B antibodies?

INPP5B has a defined domain architecture that should inform antibody selection:

• A central 5-phosphatase domain containing two conserved motifs shared with other 5-phosphatases

• A RHO GTPase-activating protein (GAP)-like domain at the C-terminus

• A prenylation sequence at the extreme C-terminus (CNPL) in mammalian INPP5B

• A linker region between these domains that mediates RAB protein interactions

Researchers should consider which domain is most suitable for their application. Antibodies targeting the 5-phosphatase domain may be useful for functional studies, while those targeting unique regions that differ from OCRL1 would be valuable for distinguishing between these related proteins.

What controls are essential for INPP5B antibody-based experiments?

Rigorous controls are critical for antibody-based studies of INPP5B:

• Include INPP5B knockout or knockdown samples as negative controls

• Use catalytically inactive INPP5B (D524A) to distinguish between enzymatic and scaffolding functions

• Employ RAB-binding deficient mutants (S589P, G688D) when studying localization dependencies

• Include domain deletion constructs to map functional regions

• Compare with closely related 5-phosphatases (particularly OCRL1) to assess specificity

• For immunoprecipitation experiments, include isotype-matched control antibodies

• In B cell experiments, include appropriate B cell receptor stimulation controls

How can researchers use INPP5B antibodies to study its role in membrane trafficking?

INPP5B plays important roles in membrane trafficking pathways, particularly in the early secretory pathway. Methodological approaches include:

• Combine INPP5B antibodies with trafficking markers (ERGIC53, GM130) for colocalization studies

• Use temperature blocks (15°C) to accumulate trafficking intermediates and assess INPP5B's effects on markers like ERGIC53

• Study INPP5B localization in cells treated with trafficking inhibitors (e.g., brefeldin A)

• Design cargo trafficking assays to measure INPP5B-dependent transport rates

• Implement live-cell imaging with fluorescently-tagged INPP5B constructs complemented by antibody validation

• Perform subcellular fractionation followed by Western blotting with INPP5B antibodies

Research has shown that expression of INPP5B alters the distribution of the cycling protein ERGIC53 at low temperature (15°C) or in the presence of brefeldin A, causing ERGIC53 to accumulate in the ERGIC with concomitant loss from the ER . This effect requires the C-terminal region of INPP5B and its interaction with RAB proteins, but not its catalytic activity .

What approaches can reveal INPP5B-dependent regulation of phosphoinositide metabolism?

To study INPP5B's enzymatic function in phosphoinositide metabolism:

• Combine INPP5B antibodies with phosphoinositide antibodies for colocalization studies

• Use phosphoinositide-specific biosensors to visualize changes in PI(4,5)P2 and PI(3,4,5)P3 levels

• Perform lipidomic analysis of membrane fractions after INPP5B manipulation

• Compare effects of wild-type INPP5B versus catalytically inactive mutant (D524A)

• Implement acute depletion systems (e.g., auxin-inducible degron) to observe immediate effects on phosphoinositide levels

• Design in vitro enzymatic assays using immunoprecipitated INPP5B to directly measure 5-phosphatase activity

These approaches can help distinguish between INPP5B's catalytic and scaffolding functions, as demonstrated by studies showing that certain effects on ERGIC53 distribution are independent of its catalytic activity .

How can researchers study INPP5B's role in B cell receptor signaling?

Recent research has identified INPP5B as a key regulator of actin remodeling, BCR clustering, and downstream signaling in antigen-stimulated B cells . Methodological approaches include:

• Use rapid auxin-induced degradation systems to acutely deplete INPP5B and observe effects on BCR clustering

• Perform time-course experiments combining INPP5B antibodies with BCR visualization to track dynamic changes during receptor clustering

• Apply live-cell microscopy to monitor BCR cap formation in control versus INPP5B-depleted cells

• Combine with actin cytoskeleton markers to study INPP5B's role in actin remodeling during BCR activation

• Use phospho-specific antibodies to monitor downstream signaling events following INPP5B depletion

• Implement proximity ligation assays to detect direct interactions with BCR components

Research has demonstrated that INPP5B regulates BCR clustering through its control of PI(4,5)P2 levels, which affects actin severing by cofilin and membrane attachment by ezrin . B cells depleted of INPP5B show impaired BCR cap formation, with approximately 70% failing to form proper caps compared to control cells .

How can researchers investigate INPP5B interactions with RAB proteins?

The interaction between INPP5B and RAB proteins is crucial for its proper localization and function . Methodological approaches include:

• Perform co-immunoprecipitation with INPP5B antibodies followed by RAB protein detection

• Use proximity ligation assays to detect direct interactions in situ

• Combine immunofluorescence microscopy with INPP5B and RAB-specific antibodies

• Compare localization of wild-type INPP5B versus RAB-binding deficient mutants (S589P and G688D)

• Use constitutively active RAB mutants (e.g., RAB5Q79L, RAB6Q72L) to enhance interactions

Research has shown that INPP5B binds to specific RAB proteins in the secretory pathway, including RAB1, RAB2, RAB5, and RAB6 . Mutation of conserved residues in the linker domain (S589P and G688D) abolishes RAB binding and results in loss of Golgi targeting, indicating that RAB interaction is required for proper INPP5B localization .

What methodologies can distinguish between INPP5B and OCRL1 functions?

Despite their similarities, INPP5B and OCRL1 have distinct functions . To differentiate between them:

• Use highly specific antibodies that do not cross-react between the two proteins

• Perform comparative localization studies using markers for different Golgi compartments (GM130 for cis-Golgi where INPP5B is enriched, TGN46 for trans-Golgi where OCRL1 is more abundant)

• Study differential binding partners (e.g., INPP5B binds RAB2 while OCRL1 does not)

• Compare effects on trafficking markers (INPP5B expression affects ERGIC53 distribution at 15°C while OCRL1 does not)

• Analyze differential interactions with clathrin and α-adaptin (present in OCRL1 but absent in INPP5B)

• Implement simultaneous knockdown/knockout experiments to assess compensatory mechanisms

What are the optimal conditions for immunoprecipitating INPP5B?

For successful immunoprecipitation of INPP5B:

• Select antibodies that recognize native epitopes rather than denatured proteins

• Consider the membrane association of INPP5B through its C-terminal prenylation when designing lysis conditions

• Use mild detergents that maintain protein-protein interactions while effectively solubilizing membrane-associated INPP5B

• Include appropriate phosphatase inhibitors to preserve phosphorylation states

• Pre-clear lysates thoroughly to reduce non-specific binding

• Consider crosslinking approaches for capturing transient interactions

• Validate results using tagged INPP5B constructs as positive controls

The search results indicate that FLAG-tagged INPP5B constructs have been successfully immunoprecipitated using anti-FLAG antibodies , providing a useful positive control system.

How should researchers design experiments to study INPP5B's role in actin remodeling?

INPP5B regulates actin remodeling through its control of PI(4,5)P2 levels . Experimental approaches include:

• Compare actin cytoskeleton organization in control versus INPP5B-depleted cells

• Analyze effects of INPP5B manipulation on actin-binding proteins regulated by PI(4,5)P2, particularly cofilin and ezrin

• Perform time-course experiments to track dynamic changes in actin organization during BCR stimulation

• Use live-cell imaging with fluorescently labeled actin to monitor remodeling in real-time

• Combine with phosphoinositide biosensors to correlate PI(4,5)P2 levels with actin changes

• Implement rescue experiments with wild-type versus catalytically inactive INPP5B

Research has demonstrated that INPP5B-dependent hydrolysis of PI(4,5)P2 promotes actin severing by cofilin and reduces actin linking to the plasma membrane by ezrin, both critical for proper BCR clustering and signaling .

What technical challenges should researchers anticipate when working with INPP5B antibodies?

Researchers should be prepared for several technical challenges:

• Potential cross-reactivity with OCRL1 due to high sequence similarity (45% identity)

• Difficulty in detecting endogenous INPP5B due to potentially low expression levels

• Variable epitope accessibility depending on INPP5B's interactions with binding partners

• Challenges in preserving membrane localization during sample preparation

• Potential differences between overexpressed and endogenous protein localization patterns

• Species-specific differences in INPP5B structure (e.g., chicken INPP5B lacks a prenylation sequence)

To address these challenges, researchers should validate antibodies using multiple approaches and include appropriate controls in all experiments.

How might INPP5B antibodies contribute to therapeutic development for B cell malignancies?

Based on recent research showing INPP5B's role in BCR signaling , potential applications include:

• Using INPP5B antibodies to assess expression levels in B cell malignancies with aberrant BCR signaling

• Screening for correlations between INPP5B expression/activity and disease progression or treatment response

• Developing activity-based probes to monitor INPP5B enzymatic function in patient samples

• Evaluating INPP5B as a potential therapeutic target for malignancies driven by dysregulated BCR signaling

• Studying compensatory mechanisms between INPP5B and related phosphatases in response to targeted therapies

Research suggests that INPP5B "may represent an attractive target for treatment of B cell malignancies caused by aberrant BCR signaling" , highlighting the potential clinical relevance of these studies.

What emerging technologies could enhance INPP5B antibody-based research?

Cutting-edge approaches that could advance INPP5B research include:

• Super-resolution microscopy to precisely map INPP5B localization relative to cellular compartments

• CRISPR/Cas9-mediated endogenous tagging to visualize INPP5B without overexpression artifacts

• Optogenetic control of INPP5B activity to study temporal aspects of its function

• Single-molecule tracking to analyze INPP5B dynamics during cellular processes

• Cryo-electron microscopy to elucidate INPP5B's structural interactions with binding partners

• Spatial transcriptomics and proteomics to map INPP5B expression and activity patterns in tissues

These technologies could help resolve current knowledge gaps and provide more detailed insights into INPP5B's cellular functions.

How can researchers quantitatively assess INPP5B enzymatic activity in different cellular contexts?

To measure INPP5B's 5-phosphatase activity:

• Develop malachite green assays to quantify phosphate release from substrate hydrolysis

• Implement fluorescence-based assays using synthetic phosphoinositide substrates

• Design HPLC methods to separate and quantify reaction products

• Compare activity of immunoprecipitated INPP5B from different cellular contexts

• Develop cell-based assays using phosphoinositide biosensors as readouts for INPP5B activity

• Engineer FRET-based biosensors to monitor INPP5B activity in real-time

These approaches would allow researchers to correlate INPP5B's enzymatic activity with its cellular functions in different contexts.