ORP4B Antibody

What is ORP4 and why is it an important target for antibody development?

ORP4 (oxysterol-binding protein-related protein 4) is a member of the OSBP family that plays essential roles in cell proliferation and survival pathways. Unlike other family members, ORP4 has been identified as essential for the survival of rapidly proliferating cells, making it a critical target for research in cancer biology and cell signaling . Antibodies against ORP4 are valuable research tools that allow for the detection, localization, and functional analysis of this protein in various experimental contexts. The significance of ORP4 is highlighted by findings showing that silencing ORP4 triggers growth arrest and apoptosis in multiple cell types, suggesting its potential role as a therapeutic target .

What are the known variants of ORP4 and how do they differ structurally?

Research has identified at least three distinct variants of ORP4:

• ORP4L: The full-length variant (approximately 95kDa) that contains an N-terminal pleckstrin homology (PH) domain, followed by the oxysterol-binding domain (OHD).

• ORP4S: A truncated variant (approximately 52kDa) that lacks the N-terminal PH domain.

• ORP4M: A 743-amino acid protein (approximately 85kDa) with a partial PH domain truncation, confirmed by PCR amplification and sequencing in HEK293 and HeLa cells .

These structural differences are significant as they likely influence the proteins' subcellular localization and functional properties. When developing or selecting antibodies, researchers must consider which variant(s) they intend to detect, as antibodies targeting different epitopes may recognize specific variants selectively or all variants collectively .

How does ORP4 function differ from other OSBP family members?

Unlike OSBP, which distributes between the endoplasmic reticulum and Golgi apparatus and mediates sterol-dependent ceramide transport, ORP4L does not localize to the Golgi apparatus in response to sterols and does not affect sphingolipid regulation . Instead, the ORP4 oxysterol-binding domain (OHD) binds to vimentin in vitro, and when overexpressed in cells, interacts with and reorganizes the vimentin network, causing it to "bundle" . Additionally, while silencing OSBP has no apparent effect on cell proliferation or survival, silencing ORP4 triggers growth arrest in some cell types (HEK293, HeLa) and apoptosis in others (IEC-18), indicating distinct functional roles between these related proteins .

What are the lipid-binding properties of ORP4 and how might this influence antibody selection?

ORP4 demonstrates dual lipid-binding properties that are crucial for its function:

• Sterol binding: ORP4 can bind oxysterols such as 25-hydroxycholesterol (25OH), which can competitively inhibit the binding of cytotoxic natural products called ORPphilins .

• Phosphoinositide binding: ORP4 has been shown to bind phosphatidylinositol 4-phosphate (PI-4P), which may be essential for its cellular functions .

When selecting antibodies for studies involving ORP4's lipid-binding functions, researchers should consider:

• Whether the antibody's epitope overlaps with the lipid-binding domains

• If the antibody binding might be affected by the presence of bound lipids

• Whether the antibody can recognize ORP4 in both lipid-bound and unbound states

This is particularly important for experiments investigating the relationship between ORP4's lipid-binding activities and its role in cell proliferation and survival .

How does ORP4 expression relate to cellular transformation and cancer biology?

Research has revealed important connections between ORP4 and cellular transformation:

• Non-transformed intestinal epithelial cells (IEC-18) undergo apoptosis characterized by caspase 3 and poly(ADP-ribose) polymerase processing, DNA cleavage, and JNK phosphorylation when ORP4 is silenced .

• The same cells transformed with oncogenic H-Ras demonstrate increased expression of ORP4L and ORP4S proteins and become resistant to the growth-inhibitory effects of ORP4 silencing .

• ORP4 appears to promote the survival of rapidly proliferating cells, suggesting it may play a role in maintaining cancer cell viability .

These findings suggest that ORP4 antibodies could be valuable tools in cancer research for:

• Assessing ORP4 expression levels in different cancer types

• Investigating the correlation between ORP4 expression and tumor aggressiveness

• Studying the mechanistic relationship between oncogenic transformations and ORP4-dependent survival pathways

What is the relationship between ORP4 and cytoskeletal components?

ORP4's interaction with the cytoskeleton, particularly with vimentin, represents a unique aspect of its cellular function. When overexpressed in cells, ORP4's oxysterol-binding domain (OHD) interacts with and collapses or "bundles" the vimentin network . While the functional relevance of this ORP4-vimentin interaction remains unclear, it aligns with observations that other ORPs and VAP also bind and affect the activity of actin and microtubule cytoskeleton components .

For researchers studying these interactions, antibodies that do not interfere with the ORP4-vimentin binding interface would be ideal for co-immunoprecipitation experiments. Additionally, antibodies suitable for immunofluorescence microscopy would be valuable for visualizing the co-localization and structural relationship between ORP4 and cytoskeletal components in different cellular contexts .

What are the recommended protocols for expression and purification of recombinant ORP4 for antibody validation?

Based on published protocols, researchers can follow these steps for recombinant ORP4 production:

• Amplify cDNAs for ORP4 variants (ORP4L, ORP4S) by PCR and clone into appropriate entry vectors (e.g., pENTR/D-TOPO) .

• Verify the sequences and recombine into expression vectors containing C-terminal tags (e.g., His₆ tag for purification) .

• Transfect the constructs into insect cells (e.g., Sf21 monolayer cultures) and select with appropriate antibiotics (e.g., ganciclovir) .

• Transduce cells with high-titer viral stocks (>10⁷ plaque-forming units/ml) at a 0.1-0.2 multiplicity of infection for 72 hours .

• Purify ORP4 from cell supernatants by metal affinity chromatography following established protocols similar to those used for OSBP .

This purified recombinant protein can then be used for antibody validation through Western blotting, ELISA, and other immunoassays to confirm specificity and sensitivity before application in complex biological samples .

What experimental approaches are most effective for studying ORP4's phosphoinositide binding properties?

To study ORP4's interaction with phosphoinositides, particularly PI-4P, researchers can employ several complementary approaches:

• Protein-lipid overlay assays: Spot phosphatidylinositol (PI) and its phosphorylated species (300 pmol), along with controls like phosphatidylcholine (PC) and solvent, onto nitrocellulose membranes. Incubate with purified ORP4 variants (50-100 nM) and detect binding using anti-ORP4 antibodies followed by fluorophore-conjugated secondary antibodies .

• Liposome binding assays: Prepare liposomes containing various percentages of phosphoinositides and assess ORP4 binding through co-sedimentation or fluorescence-based approaches.

• Surface plasmon resonance: Measure real-time binding kinetics between immobilized phosphoinositides and flowing ORP4 protein.

• Cellular localization studies: Use fluorescently tagged ORP4 in conjunction with PI-4P biosensors to monitor co-localization in live cells under various conditions.

These methodologies can be complemented with ORP4-specific antibodies for Western blotting, immunoprecipitation, or immunofluorescence to correlate binding properties with cellular functions .

How can researchers effectively use ORP4 antibodies to distinguish between protein variants in experimental samples?

To differentiate between ORP4 variants (ORP4L, ORP4M, ORP4S) in experimental samples, researchers should consider these approaches:

• Strategic antibody selection: Choose antibodies raised against epitopes that are:

  • Present in all variants (e.g., C-terminal region) for detecting total ORP4

  • Unique to specific variants (e.g., N-terminal PH domain for ORP4L) for selective detection

• Western blot analysis: Use SDS-PAGE conditions that provide good separation in the 50-100 kDa range to distinguish between ORP4L (~95 kDa), ORP4M (~85 kDa), and ORP4S (~52 kDa) .

• Immunoprecipitation coupled with mass spectrometry: For complex samples or when antibody specificity is limited, use antibodies to immunoprecipitate ORP4 followed by mass spectrometry to identify specific variants.

• RT-PCR controls: Include RT-PCR analysis with variant-specific primers as complementary validation to confirm the presence of different ORP4 transcript variants.

A systematic approach combining these methods provides the most reliable differentiation between ORP4 variants, particularly in cell types that may express multiple forms simultaneously .

What are the critical considerations for using ORP4 antibodies in RNAi experiments?

When using ORP4 antibodies to validate RNAi experiments targeting ORP4, researchers should address these important considerations:

• Variant specificity: Design siRNAs to target either specific ORP4 variants or all variants collectively. Use variant-specific antibodies to confirm the knockdown pattern aligns with the siRNA design .

• Knockdown validation: Employ antibodies in Western blots to quantitatively assess the degree of protein reduction following RNAi treatment. Aim for at least 70-80% reduction to observe functional effects .

• Phenotypic correlation: When studying growth arrest or apoptosis following ORP4 silencing, use antibodies to correlate the level of knockdown with the severity of the observed phenotype across different cell types .

• Rescue experiments: For specificity controls, perform rescue experiments with RNAi-resistant ORP4 constructs and use antibodies to confirm the expression of the rescue construct.

• Cell type considerations: Be aware that different cell types (e.g., HEK293, HeLa, IEC-18) may show different responses to ORP4 silencing (growth arrest vs. apoptosis), and antibody detection may need to be optimized for each cell type .

This methodical approach helps ensure that observed phenotypes are specifically due to ORP4 reduction rather than off-target effects .

How can researchers investigate the relationship between ORP4 and cell survival pathways?

To investigate ORP4's role in cell survival pathways, researchers can employ several antibody-dependent approaches:

• Apoptosis marker detection: Following ORP4 silencing, use antibodies against apoptotic markers such as cleaved caspase 3, processed poly(ADP-ribose) polymerase, and phosphorylated JNK to characterize the cell death mechanism .

• Signaling pathway analysis: Use phospho-specific antibodies to key signaling molecules to determine which pathways are altered when ORP4 is depleted or overexpressed.

• Co-immunoprecipitation studies: Employ ORP4 antibodies to identify interacting partners in survival signaling complexes through pull-down experiments followed by mass spectrometry or Western blotting.

• Subcellular fractionation: Use antibodies to track ORP4 localization during apoptotic events to determine if translocation occurs as part of the cell death process.

• Comparison across cell types: Apply these techniques in both normal and transformed cell lines (such as IEC-18 cells with and without H-Ras transformation) to understand how oncogenic changes affect ORP4-dependent survival mechanisms .

This multi-faceted approach can help delineate the specific mechanisms by which ORP4 promotes cell survival in different cellular contexts .

What methods can be used to study the potential therapeutic targeting of ORP4 in cancer?

Given ORP4's role in promoting the survival of rapidly proliferating cells and its increased expression in Ras-transformed cells, researchers can explore its potential as a therapeutic target using these methods:

• Expression analysis in tumor samples: Use validated ORP4 antibodies for immunohistochemistry on tissue microarrays to assess ORP4 expression across various cancer types and correlate with clinical outcomes.

• Drug screening systems: Develop cell-based assays using ORP4 antibodies to identify compounds that modulate ORP4 expression, localization, or function.

• Inhibitor mechanism studies: For compounds like ORPphilins (cephalostatin, OSW-1, ritterazine B, and schweinfurthin A) that competitively inhibit 25-hydroxycholesterol binding to ORP4L, use antibodies to assess how these compounds affect ORP4's interactions with other cellular components .

• Combination therapy assessment: Investigate how ORP4-targeting approaches might synergize with other cancer therapies by monitoring ORP4 levels and localization during treatment.

• Resistance mechanism exploration: In therapy-resistant models, use ORP4 antibodies to determine if alterations in ORP4 expression or function contribute to the resistant phenotype.

Such studies may provide insights into ORP4's potential as a novel target for anticancer drug development, particularly in contexts where its expression is elevated or required for tumor cell survival .

What are common challenges in ORP4 antibody experiments and how can they be addressed?

Researchers working with ORP4 antibodies may encounter several technical challenges:

• Cross-reactivity with other OSBP family members: Due to sequence similarities, antibodies may recognize multiple OSBP-related proteins. Solution: Validate antibody specificity using recombinant proteins and samples from ORP4-knockdown cells .

• Variant detection inconsistency: Antibodies may recognize some ORP4 variants but not others. Solution: Use multiple antibodies targeting different epitopes and compare with transcript analysis .

• Weak signal in immunofluorescence: The native expression level of ORP4 may be low in some cell types. Solution: Optimize fixation methods (paraformaldehyde vs. methanol), try antigen retrieval techniques, and use signal amplification systems.

• Background in immunoprecipitation: Non-specific binding can complicate co-immunoprecipitation studies. Solution: Optimize washing conditions and use more stringent buffers for non-specific interactions while maintaining specific ones.

• Variable results across cell types: ORP4 function and expression differ between cell types (e.g., HEK293 vs. IEC-18) . Solution: Always include appropriate positive and negative controls specific to each cell type being studied.

Addressing these challenges through rigorous validation and optimization will improve the reliability of ORP4 antibody-based experiments.

How should researchers interpret conflicting data from different ORP4 antibodies?

When faced with conflicting results from different ORP4 antibodies, researchers should follow this systematic approach:

• Epitope mapping: Determine the specific epitopes recognized by each antibody to understand if they target different domains or variants of ORP4.

• Validation hierarchy: Establish a validation hierarchy using:

  • Western blotting with recombinant ORP4 variants as positive controls

  • Samples from ORP4 knockdown experiments as negative controls

  • Orthogonal detection methods (mass spectrometry, RT-PCR) to confirm variant expression

• Binding conditions: Test whether differences in results relate to experimental conditions (denaturing vs. native, detergent types, buffer composition) that might affect epitope accessibility.

• Post-translational modifications: Consider whether conflicting results might reflect detection of differently modified forms of ORP4, which could have biological significance.

• Functional correlation: Determine which antibody results better correlate with functional readouts (e.g., growth inhibition following ORP4 silencing) .

This methodical approach can help resolve discrepancies and potentially reveal important insights about ORP4 biology that might otherwise be overlooked.