ubxn10 Antibody

What is UBXN10 and what is its biological significance in cellular functions?

UBXN10 (UBX domain-containing protein 10) is a 280 amino acid protein with a calculated molecular weight of 31 kDa that functions as a VCP/p97 binding protein required for ciliogenesis. It acts as a tethering factor that facilitates recruitment of VCP/p97 to the intraflagellar transport complex B (IFT-B) in cilia . UBX domain-containing proteins generally act as tethering factors for VCP/p97 and may specify substrate specificity of VCP/p97 .

UBXN10 is primarily enriched in the cytoplasm, suggesting it carries out its functions via translational and posttranslational modifications in this cellular compartment . Importantly, UBXN10 localizes to cilia in a VCP-dependent manner and both proteins are required for proper ciliogenesis. Live cell imaging has demonstrated GFP-UBXN10 trafficking in a bi-directional manner within cilia at rates similar to IFT88 .

What are the available types of UBXN10 antibodies and how do they differ?

UBXN10 antibodies are available in both monoclonal and polyclonal formats, each with distinct characteristics:

Monoclonal Antibodies:

• Clone OTI1H5 is a mouse monoclonal antibody available from multiple suppliers

• Concentration: 0.45 mg/mL

• Formulation: PBS with 1% BSA, 50% glycerol and 0.02% sodium azide

• Applications: Western Blot (1:500), Flow Cytometry (1:100), Immunocytochemistry/Immunofluorescence (1:100)

• Immunogen: Full-length human recombinant protein of human UBXN10 produced in HEK293T cells

Polyclonal Antibodies:

• Available from suppliers like Proteintech and Abbexa

• Host: Rabbit

• Applications: Western Blot (1:500-1:2000), Immunohistochemistry (1:50-1:500), ELISA

• Storage: -20°C, with aliquoting recommended to avoid freeze-thaw cycles

• Observed molecular weight in Western blots: 30-40 kDa

The choice between monoclonal and polyclonal depends on the specific experimental requirements, with monoclonals providing higher specificity but potentially limited epitope recognition.

What experimental applications are UBXN10 antibodies validated for?

Based on the literature and product information, UBXN10 antibodies have been validated for multiple applications:

Positive controls for WB include K-562 cells and mouse pancreas tissue. For IHC, human pancreas tissue has been validated as a positive control .

How does UBXN10 interact with the intraflagellar transport complex B (IFT-B) and what is its role in ciliogenesis?

UBXN10 interacts with the IFT-B complex through a sophisticated mechanism essential for ciliogenesis:

• Direct binding studies using GST-pulldowns with radio-labeled IFT-B subunits showed that UBXN10 associates most efficiently with CLUAP1, a component of the IFT-B complex. Weaker interactions with IFT46, IFT52, and IFT57 were also detected .

• UBXN10 functions as a tethering protein that facilitates recruitment of VCP/p97 to the IFT-B complex. Addition of HA-UBXN10 enables the association of CLUAP1 with VCP, suggesting that UBXN10 mediates this interaction .

• This interaction is specific to the IFT-B complex, as no IFT-A subunits were detected in proteomic analyses, indicating that the interaction is selective for IFT-B .

• Functionally, both VCP and UBXN10 are required for ciliogenesis. Pharmacological inhibition of VCP destabilizes the IFT-B complex and increases trafficking rates, suggesting that the VCP-UBXN10 complex plays a role in regulating the stability and function of the IFT-B complex during cilia formation .

• Localization studies have shown that UBXN10-mCHERRY and EYFP-IFT-B co-traffic in the same complex, as determined by total internal reflection fluorescence (TIRF) microscopy, further supporting their functional association .

What molecular mechanism underlies UBXN10's interaction with VCP and how does this affect its localization to cilia?

The molecular mechanism of UBXN10-VCP interaction involves a specific binding interface that is critical for UBXN10's localization to cilia:

• Structural modeling of UBXN10, based on a FAF1-VCP complex, identified a Met-Glu-Val-Pro-Arg (MEVPR) motif as the likely UBXN10-VCP interaction interface .

• Experimental validation through site-directed mutagenesis showed that mutation of each residue in this motif or deletion of the loop containing this sequence reduced binding to VCP in vivo .

• These mutations not only decreased binding to VCP but also significantly reduced the localization of GFP-UBXN10 to cilia, despite equal levels of protein expression .

• This evidence demonstrates that UBXN10 requires its interaction with VCP to properly localize within cilia, establishing a clear functional dependency on this protein-protein interaction.

• The VCP-dependency of UBXN10's ciliary localization suggests a mechanism where VCP might help transport UBXN10 to cilia or stabilize its presence within this cellular compartment.

What role does UBXN10-AS1 play in cancer progression, particularly in colon adenocarcinoma?

UBXN10-AS1 (antisense RNA 1) plays a significant tumor-suppressive role in colon adenocarcinoma (COAD) through a specific regulatory mechanism:

• Expression analysis revealed that UBXN10-AS1 and SLIT3 are expressed at low levels in COAD tissues, while miR-515-5p is expressed at high levels, suggesting their potential role in cancer pathogenesis .

• Functional studies demonstrated that UBXN10-AS1 overexpression suppresses tumor growth both in vitro and in vivo. Cell Counting Kit-8 assays showed that UBXN10-AS1 overexpression caused impaired proliferation in COAD cells, while wound healing assays proved that it impaired COAD cell migration .

• In xenograft studies, LV-UBXN10-AS1 cell-derived tumors were smaller than those in control groups and grew more slowly, confirming the anti-tumorigenic role of UBXN10-AS1 in vivo .

• Mechanistically, luciferase reporter and RNA immunoprecipitation (RIP) assays demonstrated that UBXN10-AS1 targets miR-515-5p, which in turn targets SLIT3, forming a regulatory axis .

• Further functional studies showed that miR-515-5p overexpression reversed the inhibition of COAD cell proliferation and migration caused by UBXN10-AS1 overexpression, while SLIT3 overexpression counteracted the oncogenicity of miR-515-5p .

• This UBXN10-AS1/miR-515-5p/SLIT3 regulatory network demonstrates how UBXN10-AS1 exerts its tumor-suppressive effects through modulating downstream targets involved in cell proliferation and migration.

How does UBXN10 expression vary across different tumor types according to bioinformatic analyses?

Bioinformatic analyses from databases such as TCGA have revealed distinct patterns of UBXN10 expression across various tumor types:

• UBXN10 expression is downregulated in the majority of tumor types compared to non-cancerous tissues, with the exceptions of kidney renal papillary cell carcinoma (KIRP) and breast invasive carcinoma (BRCA), where it shows upregulation .

• In contrast, another UBXD family member, ASPSCR1, shows upregulation in most tumors except for kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), and thyroid carcinoma (THCA) .

• All UBXD family members, including UBXN10, were found to be downregulated in kidney chromophobe (KICH), suggesting a potential common regulatory mechanism in this tumor type .

• Immunohistochemical analyses from the Human Protein Atlas showed that UBXN10 protein typically exhibits weak to moderate cytoplasmic and/or nuclear immunoreactivity in most cancer tissues .

• Specific cancer types show variable staining patterns: weak or negative staining has been observed in some hepatocellular carcinoma, endometrial carcinoma, and kidney cancer samples, suggesting tissue-specific regulation of UBXN10 expression .

• These differential expression patterns across tumor types suggest potential tissue-specific functions of UBXN10 in cancer development and progression.

How should researchers optimize Western blot protocols for UBXN10 detection?

For optimal Western blot detection of UBXN10, researchers should consider the following technical parameters and controls:

• Sample Preparation:

  • Use appropriate lysis buffers that preserve protein integrity

  • Include protease inhibitors to prevent degradation

  • K-562 cells and mouse pancreas tissue are verified positive controls

• Protein Loading and Transfer:

  • Load 20-50 μg of total protein per lane

  • Use a 10-12% polyacrylamide gel for optimal resolution in the 30-40 kDa range

  • Transfer to PVDF or nitrocellulose membranes at 100V for 1-2 hours or 20-30V overnight

• Antibody Selection and Dilution:

  • For monoclonal antibodies: use at 1:500 dilution

  • For polyclonal antibodies: use at 1:500-1:2000 dilution

  • Incubate primary antibody overnight at 4°C for best results

• Expected Results and Validation:

  • Expect bands at 30-40 kDa molecular weight range

  • Include positive controls (K-562 cells, mouse pancreas tissue)

  • Include negative controls and loading controls for normalization

  • Consider using UBXN10-overexpressing cells (like transfected SW480 cells) as additional positive controls

• Troubleshooting Common Issues:

  • For high background: increase blocking time or washing steps

  • For weak signal: increase antibody concentration or protein loading

  • For multiple bands: optimize antibody dilution or consider using a more specific antibody

What are the critical considerations for immunohistochemical detection of UBXN10?

For successful immunohistochemical detection of UBXN10, researchers should follow these critical guidelines:

• Tissue Preparation and Fixation:

  • Use freshly fixed tissues when possible

  • Standard formalin fixation and paraffin embedding are commonly employed

  • Optimal section thickness is typically 4-6 μm

• Antigen Retrieval (Critical Step):

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: citrate buffer pH 6.0

  • Heating conditions: usually 95-98°C for 15-20 minutes

• Antibody Selection and Dilution:

  • Recommended dilution range: 1:50-1:500 for IHC

  • Incubation time: typically overnight at 4°C or 1-2 hours at room temperature

  • Sensitivity can vary by tissue type and fixation method

• Positive and Negative Controls:

  • Human pancreas tissue is a verified positive control

  • Include isotype controls to detect non-specific binding

  • Adjacent normal tissue can serve as internal controls when available

• Expected Staining Patterns:

  • UBXN10 typically shows weak to moderate cytoplasmic and/or nuclear immunoreactivity in most cancer tissues

  • Staining intensity varies by cancer type, with some tumors showing stronger positivity than others

  • Some hepatocellular carcinoma, endometrial carcinoma, and kidney cancer samples may show weak positive or negative staining

• Optimization Parameters:

  • Titrate antibody concentration based on tissue type

  • Adjust incubation times and temperatures as needed

  • Consider signal amplification for low-expressing tissues

How can researchers validate the specificity of UBXN10 antibodies in their experimental system?

Comprehensive validation of UBXN10 antibodies is essential for reliable experimental results. Researchers should implement the following validation strategies:

• Multi-method Verification:

  • Compare results across different applications (WB, IHC, ICC) when possible

  • Verify that molecular weight in Western blots matches the expected 30-40 kDa range

  • Confirm that subcellular localization in ICC/IF matches expected cytoplasmic pattern or ciliary localization in appropriate cell types

• Positive and Negative Controls:

  • Use verified positive controls:

    • K-562 cells and mouse pancreas tissue for Western blot

    • Human pancreas tissue for IHC

    • Cells with known cilia for ICC/IF of ciliary UBXN10 (e.g., hTERT-RPE1, IMCD, or LLC-PK1 cells)

  • Include appropriate negative controls:

    • Isotype controls for immunostaining

    • Samples known not to express UBXN10

• Genetic Validation Approaches:

  • Overexpression system: transfect cells with UBXN10 expression vectors and verify increased signal

  • Knockdown/knockout validation: use siRNA or CRISPR to reduce UBXN10 expression and confirm decreased signal

  • Tagged protein validation: express tagged UBXN10 and confirm co-localization with antibody staining

• Cross-reactivity Assessment:

  • Peptide competition assay: pre-incubate antibody with immunizing peptide to block specific binding

  • Cross-species validation: test reactivity with human and mouse samples as indicated by product information

  • Test antibody on protein arrays when available - some UBXN10 antibodies have been tested on arrays of 364 human recombinant protein fragments

• Reproducibility Testing:

  • Test multiple antibody lots when available

  • Compare results from different antibody clones or suppliers

  • Document all validation experiments thoroughly for reproducibility

What are the recommended protocols for studying UBXN10's interaction with the IFT-B complex?

To investigate UBXN10's interaction with the IFT-B complex, researchers should consider these experimental approaches based on published methodologies:

• Co-immunoprecipitation Assays:

  • Immunoprecipitate UBXN10 and blot for IFT-B components (especially CLUAP1, IFT46, IFT52, and IFT57)

  • Reciprocal IP: immunoprecipitate IFT-B components (e.g., IFT88, IFT74, TTC26) and blot for UBXN10

  • Include VCP in the analysis as it forms part of the UBXN10-IFT-B complex

  • Suggested lysis buffer: standard IP buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40 or Triton X-100, and protease inhibitors

• GST-Pulldown Assays for Direct Binding:

  • Express and purify GST-UBXN10 from E. coli

  • Incubate with in vitro translated radio-labeled IFT-B subunits

  • Focus particularly on CLUAP1, which shows the strongest direct interaction

  • Include GST alone as a control to identify non-specific binding

• In Vitro Translation Systems:

  • Co-translate MYC-tagged IFT-B subunits with HA-UBXN10 in a HeLa extract translation system

  • Perform co-immunoprecipitation to test interactions

  • This system allows extract-derived IFT-B subunits to assemble with the translated subunit

• Live Cell Imaging for Co-trafficking:

  • Express fluorescently tagged proteins (e.g., GFP-UBXN10 and fluorescently tagged IFT-B components)

  • Use appropriate ciliated cell lines: hTERT-RPE1, IMCD, or LLC-PK1 cells

  • Perform live cell imaging using TIRF microscopy to observe co-trafficking

  • Analysis parameters: trafficking rates should be similar to IFT88 (a well-characterized IFT-B component)

• Structure-Function Analysis:

  • Create UBXN10 mutants with alterations in key domains or motifs (e.g., MEVPR motif)

  • Test mutants' ability to bind VCP and IFT-B components

  • Examine the effect on ciliary localization and ciliogenesis

  • Use this approach to map critical interaction interfaces

What considerations are important when designing experiments to study UBXN10's role in cancer?

When investigating UBXN10's role in cancer, researchers should consider these experimental design principles:

• Expression Analysis Approaches:

  • Analyze UBXN10 and UBXN10-AS1 expression across multiple cancer types using qRT-PCR

  • Compare matched tumor and adjacent normal tissues to identify differential expression

  • Consider analyzing additional genes in the regulatory network, including miR-515-5p and SLIT3

  • Follow established protocols: for RT-qPCR of UBXN10-AS1 in COAD studies, researchers used SW480 and SW620 cells with significant results

• Functional Assay Selection:

  • Proliferation: Cell Counting Kit-8 assays showed that UBXN10-AS1 overexpression impaired proliferation in COAD cells

  • Migration: Wound healing scratch assays demonstrated that UBXN10-AS1 overexpression impaired COAD cell migration

  • In vivo tumorigenesis: Xenograft models using BALB/c mice with subcutaneous injection of modified cells (e.g., SW480 cells infected with LV-UBXN10-AS1 vectors)

• Molecular Mechanism Investigations:

  • Luciferase reporter assays to detect direct interactions between UBXN10-AS1, miR-515-5p, and SLIT3

  • RNA immunoprecipitation (RIP) assays to confirm RNA-protein interactions

  • Rescue experiments: test whether miR-515-5p overexpression reverses the effects of UBXN10-AS1 overexpression, and whether SLIT3 overexpression counteracts miR-515-5p effects

• Model System Considerations:

  • Cell lines: SW480 and SW620 colon cancer cell lines have been successfully used in UBXN10-AS1 studies

  • Animal models: BALB/c nude mice have been used for xenograft studies with UBXN10-AS1-overexpressing cells

  • Sample size and power calculations: ensure sufficient statistical power for detecting biologically meaningful differences

• Clinical Correlation Analysis:

  • Correlate UBXN10/UBXN10-AS1 expression with clinical parameters (stage, grade, survival)

  • Consider analyzing TCGA datasets, which have shown UBXN10 downregulation in multiple tumor types

  • Evaluate potential as a prognostic biomarker, as some UBXD family members have been linked to survival and cancer progression