INPP5B Antibody, FITC conjugated

What is INPP5B and what are its primary cellular functions?

INPP5B is a 75 kDa inositol polyphosphate-5-phosphatase that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PtIns(4,5)P2) and phosphatidylinositol 1,4,5-trisphosphate (PtIns(1,4,5)P3), thereby modulating cellular signaling events . INPP5B is a key regulator of actin remodeling, B cell receptor (BCR) clustering, and downstream signaling in antigen-stimulated B cells. This regulation occurs through INPP5B-dependent hydrolysis of PI(4,5)P2, which controls cortical actin and B cell receptor dynamics . INPP5B is also localized to the early secretory pathway including the Golgi apparatus and ER-to-Golgi intermediate compartment (ERGIC), suggesting a potential role in membrane trafficking .

How does INPP5B differ from its homolog OCRL1?

While INPP5B shares similar substrate specificity and domain organization with the Lowe syndrome protein OCRL1, they exhibit distinct functional characteristics. Unlike OCRL1, INPP5B does not interact with clathrin or α-adaptin and is largely absent from clathrin-coated intermediates . The function of INPP5B in BCR clustering and cell spreading is not shared with OCRL1 . Additionally, INPP5B expression affects the distribution of the cycling protein ERGIC53 in ways that OCRL1 does not, suggesting a unique role for INPP5B in retrograde ERGIC-to-ER transport . Despite these differences, INPP5B can compensate for loss of OCRL1 in knockout mice, highlighting their functional overlap in certain contexts .

What are the established applications for INPP5B antibodies in research?

INPP5B antibodies are primarily used in Western blotting for detecting endogenous INPP5B protein, with effectiveness demonstrated in human samples . While standard antibodies are valuable for protein detection, FITC-conjugated variants offer additional applications in immunofluorescence studies, particularly for investigating INPP5B localization within the secretory pathway and at the B cell receptor signalosome. These antibodies can be instrumental in studying INPP5B's role in actin remodeling during B cell activation, where researchers can visualize the spatial and temporal dynamics of INPP5B in relation to the cytoskeleton and BCR clusters.

How do FITC-conjugated INPP5B antibodies facilitate complex B cell signaling studies?

FITC-conjugated INPP5B antibodies provide a powerful tool for visualizing the dynamics of INPP5B during B cell receptor activation. When designing experiments to investigate BCR signaling, researchers should consider the following methodology:

• Use surface-bound anti-IgM antibodies as surrogate antigen to stimulate B cells

• Apply FITC-conjugated INPP5B antibodies to track INPP5B redistribution during BCR clustering

• Implement TIRF microscopy to visualize events at the plasma membrane interface

• Incorporate phalloidin staining to simultaneously visualize F-actin remodeling

This approach enables analysis of how INPP5B regulates actin dynamics and BCR clustering in response to antigen stimulation . Quantitative metrics should include BCR cluster counts, cell spreading area, and colocalization coefficients between INPP5B and BCR clusters.

What experimental considerations are important when using INPP5B antibodies for phosphoinositide metabolism studies?

When investigating INPP5B's role in phosphoinositide metabolism, researchers should design experiments that:

• Track multiple phosphoinositide species simultaneously

• Account for the specific subcellular localization of INPP5B

• Consider temporal dynamics of PI(4,5)P2 and PI(3,4,5)P3 hydrolysis

The established degradation system for endogenous INPP5B protein used in the DT40 cell line provides an excellent model for studying these dynamics . This system demonstrated that INPP5B depletion results in significant decreases in PI(3,4,5)P3 abundance following BCR stimulation, consistent with reduced PI3K signaling . When using FITC-conjugated INPP5B antibodies in such studies, consider dual labeling with PI(4,5)P2-specific probes to correlate INPP5B localization with its substrate.

How can researchers investigate INPP5B's role in retrograde transport using FITC-conjugated antibodies?

To study INPP5B's function in retrograde ERGIC-to-ER transport, implement the following methodology:

• Utilize temperature manipulation (15°C incubation) to slow transport between compartments

• Apply brefeldin A treatment to examine ERGIC53 distribution patterns

• Use FITC-conjugated INPP5B antibodies to track enzyme localization

• Compare INPP5B localization with markers for ERGIC and Golgi compartments

Experimental evidence indicates that expression of INPP5B alters the distribution of cycling protein ERGIC53 under these conditions, causing ERGIC53 to accumulate in the ERGIC with concomitant loss from the ER . This suggests INPP5B plays a role in retrograde transport distinct from that of OCRL1.

What are the optimal conditions for INPP5B detection by Western blotting?

For optimal Western blot results with INPP5B antibodies, follow these technical parameters:

These conditions have been validated for unlabeled INPP5B antibodies . When using FITC-conjugated variants, additional optimization may be necessary, particularly regarding exposure settings to account for the fluorescent signal. Consider running a dilution series (1/500-1/2000) to determine optimal signal-to-noise ratio for your specific experimental system.

What controls should be included when validating INPP5B antibody specificity?

To ensure specificity of FITC-conjugated INPP5B antibodies, incorporate these critical controls:

• Knockdown/knockout validation: Utilize INPP5B-depleted cells as negative controls, such as the auxin-inducible degradation system described in DT40 cells

• Competitive inhibition: Pre-incubate antibody with purified INPP5B protein before application

• Cross-reactivity assessment: Test antibody against related inositol 5-phosphatases, particularly OCRL1

• Secondary antibody-only control: Exclude primary antibody to identify non-specific binding

• Isotype control: Use FITC-conjugated antibodies of the same isotype but different specificity

This comprehensive validation approach ensures that observed signals are specific to INPP5B rather than artifacts from non-specific binding.

How do fixation and permeabilization conditions affect FITC-conjugated INPP5B antibody performance?

The choice of fixation and permeabilization methods can significantly impact FITC-conjugated INPP5B antibody performance in immunofluorescence studies:

• Paraformaldehyde fixation (4%): Preserves cytoskeletal structures but may reduce epitope accessibility

• Methanol fixation: Enhances detection of certain epitopes but disrupts membrane structures

• Mild detergent permeabilization (0.1% Triton X-100): Balances epitope accessibility with structural preservation

• Saponin permeabilization (0.1%): Preferred for studying membrane-associated proteins like INPP5B

Consider that INPP5B associates with membrane compartments including the Golgi apparatus and ERGIC , so fixation methods that best preserve these structures while maintaining epitope accessibility will yield optimal results.

How should experiments be designed to investigate INPP5B's interaction with Rab proteins?

To study INPP5B's interaction with Rab proteins in the secretory pathway, implement this methodological approach:

• Generate INPP5B constructs with mutations in the Rab-binding domain

• Compare localization of wild-type and mutant INPP5B using FITC-conjugated antibodies

• Perform co-immunoprecipitation studies with specific Rab proteins

• Assess functional consequences of disrupting INPP5B-Rab interactions on secretory pathway dynamics

Research has established that Rab binding is required for efficient Golgi targeting of INPP5B , making this interaction critical for understanding INPP5B's function in the secretory pathway. When designing these experiments, consider that INPP5B binds specifically to certain Rab proteins within this pathway.

What approaches can be used to study INPP5B's role in actin dynamics during B cell activation?

To investigate INPP5B's role in actin remodeling during B cell activation, employ these methodological strategies:

• Utilize FITC-conjugated INPP5B antibodies alongside actin visualization (phalloidin staining)

• Design time-course experiments capturing early events (0-5 minutes) after BCR stimulation

• Implement quantitative image analysis measuring:

  • Cortical actin density

  • Cofilin activation state (phospho-specific antibodies)

  • Ezrin recruitment to the plasma membrane

• Compare INPP5B-depleted cells with control cells during BCR activation

This approach will help elucidate how INPP5B-mediated PI(4,5)P2 hydrolysis affects actin severing by cofilin and actin linking to the plasma membrane by ezrin, both of which are sensitive to INPP5B-dependent PI(4,5)P2 changes .

How can multiplexed imaging approaches be implemented when using FITC-conjugated INPP5B antibodies?

For multiplexed imaging studies with FITC-conjugated INPP5B antibodies, consider these methodological strategies:

• Spectral compatibility: Pair FITC (excitation ~495nm, emission ~520nm) with spectrally distinct fluorophores such as:

  • Cy3 (ex: ~550nm, em: ~570nm) for BCR labeling

  • Alexa 647 (ex: ~650nm, em: ~665nm) for subcellular compartment markers

• Sequential imaging protocols: To minimize bleed-through between channels:

  • Acquire FITC signal first (as it is more susceptible to photobleaching)

  • Follow with longer-wavelength fluorophores

  • Apply appropriate background subtraction between acquisitions

• Co-staining optimization: When studying BCR dynamics alongside INPP5B:

  • Label BCR with monovalent anti-IgM Fab fragments conjugated to a red fluorophore

  • Visualize F-actin with far-red fluorophore-conjugated phalloidin

  • Use DAPI for nuclear counterstaining

This approach enables simultaneous visualization of INPP5B, BCR clustering, and actin remodeling during B cell activation, providing comprehensive insights into the spatial and temporal relationships between these components.

How might FITC-conjugated INPP5B antibodies contribute to understanding B cell malignancies?

INPP5B has been identified as a potential therapeutic target for B cell malignancies caused by aberrant BCR signaling . Research using FITC-conjugated INPP5B antibodies could:

• Compare INPP5B expression and localization patterns between normal and malignant B cells

• Correlate INPP5B levels with BCR signaling strength in various B cell lymphoma subtypes

• Assess how pharmacological inhibition of INPP5B affects malignant B cell survival and proliferation

• Investigate the relationship between INPP5B activity and resistance to current B cell lymphoma therapies

This research direction may yield valuable insights into the pathogenesis of B cell malignancies and potentially identify new therapeutic approaches targeting INPP5B-dependent signaling pathways.

What are the considerations for developing live-cell imaging approaches with INPP5B antibodies?

For live-cell imaging applications studying INPP5B dynamics:

• Consider alternative conjugates to FITC that offer greater photostability (e.g., Alexa 488)

• Implement fluorescent protein tagging strategies (GFP-INPP5B) as complementary approaches

• Develop nanobody-based detection systems that can access intracellular INPP5B without permeabilization

• Employ FRAP (Fluorescence Recovery After Photobleaching) techniques to study INPP5B mobility

When designing these experiments, consider that INPP5B localization changes during BCR stimulation, with implications for both endocytic and secretory pathway functions .

Through proper experimental design and methodological rigor, FITC-conjugated INPP5B antibodies can provide valuable insights into the complex roles of this phosphoinositide phosphatase in cellular signaling and membrane trafficking.