RANBP9 Monoclonal Antibody

What is RANBP9 and what are its primary cellular functions?

RANBP9 is a scaffolding protein that plays pivotal roles in multiple cellular processes. Research has demonstrated that RANBP9 physically interacts with amyloid precursor protein (APP), low-density lipoprotein receptor-related protein (LRP), and β-integrins, forming protein complexes that affect both Alzheimer's disease pathology and cell adhesion mechanisms. RANBP9 significantly disrupts integrin-dependent cell attachment and spreading in NIH3T3 and hippocampus-derived HT22 cells, while simultaneously decreasing Pyk2/paxillin signaling pathways and talin/vinculin localization in focal adhesion complexes . This multi-functional protein serves as a critical regulatory hub connecting protein trafficking, cell-matrix interactions, and amyloid processing pathways.

What is the molecular structure and detected weight of RANBP9?

RANBP9 is a relatively large protein with distinct structural domains including SPRY and LisH domains that mediate protein-protein interactions. Based on amino acid sequence analysis, the calculated molecular weight of RANBP9 is 78 kDa (729 amino acids), though the observed molecular weight on Western blots typically appears between 80-90 kDa due to post-translational modifications . The protein contains functional domains that enable interactions with multiple binding partners. When working with RANBP9 monoclonal antibodies, it's important to note this size discrepancy to properly identify the protein band during Western blot analyses.

What tissue distribution and expression patterns does RANBP9 exhibit?

RANBP9 demonstrates broad tissue expression with particularly notable levels in neural and reproductive tissues. Immunohistochemical analyses have identified positive detection in human testis tissue, human skin tissue, mouse testis tissue, and rat testis tissue . Additionally, Western blot analyses have confirmed RANBP9 expression in mouse brain tissue and various cell lines including HeLa cells . The widespread distribution suggests tissue-specific functions that may vary depending on the cellular context, highlighting the importance of selecting appropriate experimental models when studying RANBP9 function.

How does RANBP9 contribute to Alzheimer's disease pathogenesis?

RANBP9 plays a critical role in Alzheimer's disease pathology through several mechanisms that promote amyloid beta (Aβ) production. Co-immunoprecipitation experiments from wild-type mouse brain demonstrate that endogenous APP and LRP form complexes with endogenous RANBP9 in vivo . Stable overexpression of RANBP9 in CHO cells expressing APP751 results in approximately 3.5-fold increase in Aβ secretion compared to control cells without altering APP holoprotein levels .

The mechanism involves RANBP9 markedly increasing the secretion of sAPP-β while reducing sAPP-α without changing total sAPP levels, indicating a shift toward the amyloidogenic processing pathway . This effect occurs independently of the KPI domain, as similar results were observed in cells expressing APP695 . RANBP9 accelerates APP internalization approximately 2.3-fold relative to surface APP levels, which partially explains the enhanced β-secretase processing and increased Aβ generation .

What is the relationship between RANBP9 and focal adhesion signaling?

RANBP9 serves as a negative regulator of integrin-mediated cell adhesion and focal adhesion assembly. Experimental data shows that RANBP9 overexpression dramatically impairs cell attachment and spreading on fibronectin-coated surfaces in both NIH3T3 and HT22 cells . This inhibition occurs concurrently with decreased Pyk2/paxillin signaling and disrupted talin/vinculin localization in focal adhesion complexes .

Conversely, RanBP9 knockdown via siRNA techniques demonstrates the opposite effect, robustly promoting cell attachment and spreading, with enhanced focal adhesion signaling and assembly . These findings establish RANBP9 as a critical modulator of cellular adhesion processes, suggesting potential applications for RANBP9 antibodies in studying cell migration, wound healing, and tissue architecture maintenance.

How can RanBP9 transgenic mouse models be utilized in research?

RanBP9 transgenic mouse models provide valuable tools for studying its function in vivo. These models are typically generated by inserting N-terminally Flag-tagged human full-length RanBP9 into the mouse Thy-1 expression cassette at a unique SalI site . The construct is then microinjected into fertilized embryos from superovulated CB6 mice and implanted into pseudopregnant recipient mice .

Several transgenic lines have been established (including lines 528, 599, and 629) and maintained by crossing transgenic progeny with C57BL6 mice . Identification of transgenic mice is accomplished through PCR genotyping from tail-derived genomic DNA using specific primers that yield a 443 bp PCR product only in RanBP9-transgenic mice . These models allow researchers to investigate RANBP9's impact on neuronal development, APP processing, and Alzheimer's disease pathology in an organism-wide context.

What are the optimal conditions for Western blot detection of RANBP9?

For successful Western blot detection of RANBP9, researchers should follow specific protocol optimizations. The recommended antibody dilution range for Western blot applications is 1:1000-1:4000 . RANBP9 typically appears as a band between 80-90 kDa, which is slightly higher than its calculated molecular weight due to post-translational modifications .

Positive detection has been validated in multiple sample types including mouse brain tissue, HeLa cells, and mouse testis . For optimal results, it is recommended to titrate the antibody concentration based on the specific sample type and detection system used.

What techniques are most effective for visualizing RANBP9 in tissues and cells?

Multiple imaging techniques can effectively visualize RANBP9 distribution with appropriate antibody optimization:

For immunohistochemical applications, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may serve as an alternative . When performing immunofluorescence studies, appropriate controls should be included to confirm specificity, particularly in co-localization experiments with other focal adhesion or APP pathway components.

How can cell surface biotinylation be used to study RANBP9's effect on protein trafficking?

Cell surface biotinylation represents a powerful technique for investigating RANBP9's role in regulating surface expression and endocytosis of integral membrane proteins. The protocol involves biotinylating cell surface proteins on ice, then lysing cells and immunoprecipitating with anti-biotin antibody to isolate and analyze surface proteins by Western blotting .

For endocytosis assays, cells are surface biotinylated with a cleavable biotin derivative on ice, returned to 37°C for specific time periods (typically 3 minutes), after which non-internalized surface biotin is removed . Lysates are then immunoprecipitated for biotin and analyzed for internalized proteins. Using this approach, researchers demonstrated that RANBP9 overexpression accelerates LRP endocytosis by approximately 50% and β1-integrin endocytosis by approximately 3-fold .

What contradictions exist in RANBP9 research data and how should researchers address them?

When analyzing RANBP9 function, researchers may encounter seemingly contradictory results between in vitro overexpression systems and in vivo physiological contexts. For example, while RANBP9 overexpression dramatically disrupts cell adhesion in transfected cell lines, its endogenous function in tissues with naturally high expression (such as testis) requires careful interpretation, as these tissues maintain normal adhesive properties despite high RANBP9 levels .

To address these contradictions, comprehensive experimental approaches are recommended, including:

• Using both gain-of-function (overexpression) and loss-of-function (siRNA knockdown) approaches

• Validating findings across multiple cell types and tissue contexts

• Combining in vitro studies with in vivo models including transgenic mice

• Employing quantitative assays for cell attachment, protein trafficking, and Aβ generation

This multifaceted approach helps distinguish between physiological functions and potential artifacts from experimental manipulations.

How can researchers validate RANBP9 antibody specificity?

Validating antibody specificity is crucial for reliable RANBP9 detection and characterization. A multi-step validation approach includes:

• Western blot validation: Confirm a single band at the expected 80-90 kDa size with minimal non-specific binding

• siRNA knockdown controls: RANBP9 siRNA should reduce the signal by approximately 90% compared to control siRNA-transfected cells

• Transgenic model validation: Compare signals between wild-type and RANBP9-transgenic mice using both commercial antibodies and epitope tag detection (such as anti-Flag M2)

• Cross-reactivity testing: Verify reactivity across relevant species including human, mouse, and rat samples

This comprehensive validation protocol ensures that observed results genuinely reflect RANBP9 biology rather than artifacts from non-specific antibody binding.

What factors influence reproducibility in RANBP9 detection assays?

Multiple technical factors can affect the reproducibility of RANBP9 detection:

• Sample preparation: Extraction methods significantly impact protein recovery, with RIPA buffer recommended for most applications

• Antibody storage conditions: Store at -20°C with 0.02% sodium azide and 50% glycerol (pH 7.3) to maintain stability for one year after shipment

• Antigen retrieval methods: For IHC applications, TE buffer pH 9.0 provides optimal results compared to alternative methods

• Cell culture conditions: Transfection efficiency must be monitored (typically ~90% for NIH3T3 and HT22 cells with standard protocols)

• Signal detection sensitivity: Enhanced chemiluminescence systems may be required for detecting endogenous RANBP9 in certain tissues

By standardizing these factors across experiments, researchers can achieve more consistent and comparable results when studying RANBP9 biology.

How should researchers design experiments to investigate RANBP9's role in protein complex formation?

When investigating RANBP9's scaffolding functions in protein complex formation, carefully designed co-immunoprecipitation experiments are essential. Experimental designs should include:

• Reciprocal co-immunoprecipitations: Pull down with RANBP9 antibody and probe for interaction partners (APP, LRP, integrins), then repeat with antibodies against suspected binding partners

• Domain mapping: Utilize constructs expressing specific RANBP9 domains (SPRY-LisH) to identify regions responsible for protein interactions

• In vivo validation: Confirm interactions occur with endogenous proteins in physiologically relevant tissues such as brain samples

• Quantitative analysis: Measure relative binding affinities under different conditions to identify regulatory mechanisms

These approaches can reveal how RANBP9 simultaneously coordinates APP processing, integrin trafficking, and focal adhesion assembly to influence both Alzheimer's pathology and cell adhesion.

How might RANBP9 antibodies contribute to therapeutic target identification for Alzheimer's disease?

RANBP9 antibodies can facilitate the identification and validation of potential therapeutic targets for Alzheimer's disease through several research applications:

• Target validation studies: Using RANBP9 antibodies to monitor protein levels in patient tissues compared to controls

• Interaction disruption screening: Identifying small molecules that disrupt RANBP9-APP-BACE1 complexes using co-immunoprecipitation with RANBP9 antibodies

• Therapeutic antibody development: Using anti-RANBP9 antibodies as tools to validate the accessibility of RANBP9 epitopes in vivo

• Biomarker development: Exploring whether RANBP9 levels correlate with disease progression using sensitive detection methods

Given that RANBP9 overexpression increases Aβ production approximately 3.5-fold , interventions targeting RANBP9-dependent APP processing represent promising therapeutic avenues for reducing amyloid burden in Alzheimer's disease.

What novel applications exist for RANBP9 antibodies in neurodevelopmental research?

RANBP9 antibodies offer valuable tools for investigating neurodevelopmental processes based on RANBP9's demonstrated impact on neuronal morphology:

• Neurite outgrowth analysis: Primary hippocampal neurons from RANBP9-transgenic mice show severely reduced neurite arborization

• Synaptic development studies: Immunofluorescence applications can track RANBP9 localization during synaptogenesis

• Cell migration tracking: RANBP9's effects on integrin trafficking suggest roles in neuronal migration during development

• Protein trafficking visualization: Combining RANBP9 antibodies with APP and integrin markers can reveal developmental regulation of endocytic pathways

These applications could provide insights into both normal neurodevelopment and pathological processes in neurodevelopmental disorders with altered cell adhesion or protein trafficking components.