NUB1 Antibody

What is NUB1 and what is its primary function in cells?

NUB1 (NEDD8 Ultimate Buster-1) is a 68-74 kDa, TNF-alpha and interferon-inducible protein that functions as a specific down-regulator of the NEDD8 conjugation system. It recruits NEDD8, UBD, and their conjugates to the proteasome for degradation. NUB1 plays a critical role in regulating protein degradation pathways by targeting both free and conjugated NEDD8 to the proteasome, effectively reducing NEDD8's presence in cells .

Human NUB1 is 615 amino acids in length and contains two coiled-coil regions (amino acids 36-70 and 152-203) and three UBA domains (amino acids 374-413, 424-470, and 489-529) . There are three potential isoform variants, including one with an alternative start site and another showing specific amino acid deletions .

What applications are NUB1 antibodies commonly used for in research?

Based on validated research protocols, NUB1 antibodies are primarily used for:

These applications have been validated across multiple antibody sources and research publications .

How can I validate the specificity of NUB1 antibodies?

To validate NUB1 antibody specificity:

• Positive control selection: Use cell lines known to express NUB1, such as HEK-293, HeLa, or Raji cells. Western blot should detect a band at approximately 69-78 kDa .

• RNAi validation: Employ RNA interference (siRNA) targeting NUB1. The specific siRNA sequence reported in literature corresponds to nucleotides 458 to 476 after the start codon: 5′-CGAUGGUGCUUGAACUAAAUU-3′ and 5′-UUUAGUUCAAGCACCAUCGUU-3′. Compare antibody signal between control and NUB1-silenced samples .

• Overexpression validation: Transfect cells with FLAG-tagged NUB1 expression constructs (pcDNA3/FLAG-NUB1) and confirm increased signal with anti-NUB1 antibody compared to mock-transfected cells .

• Multiple antibody concordance: When possible, compare results from antibodies from different vendors or those targeting different epitopes of NUB1 .

How can I design experiments to study NUB1-dependent protein degradation pathways?

To investigate NUB1-dependent protein degradation pathways:

• Proteasome inhibition experiments: Treat cells with proteasome inhibitors like MG132 (15 μM, 12 hours) alongside NUB1 overexpression or knockdown. If NUB1 regulates a protein through proteasomal degradation, MG132 treatment should abolish the effect of NUB1 manipulation on target protein levels .

• Ubiquitination assay: Perform co-immunoprecipitation (Co-IP) followed by western blotting with anti-ubiquitin antibodies to detect changes in ubiquitination of target proteins when NUB1 levels are altered .

• NEDDylation analysis: Since NUB1 specifically regulates the NEDD8 pathway, analyze NEDDylation of suspected target proteins using Co-IP followed by anti-NEDD8 western blotting. Compare NEDDylation patterns between wild-type conditions and NUB1 knockdown or overexpression .

• Half-life determination: Perform cycloheximide chase assays to measure protein half-life changes in response to NUB1 manipulation, which can indicate whether NUB1 affects protein stability through degradation pathways .

A key example from hepatocellular carcinoma research shows that NUB1 reduction leads to increased PCNA NEDDylation at lysine 164, which antagonizes PCNA K48-linked polyubiquitination, thereby increasing PCNA stability .

What protocols are recommended for studying NUB1 in protein-protein interaction networks?

To investigate NUB1 protein interactions:

• Yeast two-hybrid (Y2H) assays: Follow the protocol used to study NUB1 interaction with synphilin-1:

  • Subclone synphilin-1 cDNA into pGADT7 (Gal4 DNA-activating domain vector)

  • Subclone NUB1 cDNA into pGBKT7 (Gal4 DNA-binding domain vector)

  • Co-transfect both constructs into AH109 yeast cells using the lithium acetate method

  • Grow transformed cells on His−/Trp−/Leu− synthetic agar plates for 3 days at 30°C

  • Determine specific protein-protein interaction by cell growth on selection plates

• Co-immunoprecipitation (Co-IP):

  • Prepare cell lysates in appropriate buffer

  • Incubate with anti-NUB1 antibody (5-10 μg)

  • Capture immune complexes with protein G-Sepharose

  • Wash extensively to remove non-specific binding

  • Analyze by western blotting with antibodies against suspected interaction partners

• Domain mapping: Create NUB1 mutants to identify interaction domains. For example, the NEDD8-binding site in NUB1 is critical for its interaction with synphilin-1, suggesting overlapping binding regions .

How can I design experiments to study NUB1's role in cancer cell proliferation?

Based on research findings, these methodological approaches are recommended:

• Cell proliferation assays:

  • 5-Ethynyl-2'-deoxyuridine (EdU) incorporation assay to measure DNA synthesis

  • Colony formation assay to assess long-term proliferative capacity

  • Compare results between NUB1 knockdown, overexpression, and control conditions

• In vivo tumor growth models:

  • Establish subcutaneous tumor models in BALB/c nude mice using cells with altered NUB1 expression

  • Monitor tumor growth over 45 days

  • Use in vivo imaging system (IVIS) to measure fluorescence intensity

  • Measure tumor volume and weight at endpoint

  • Analyze tumor tissue by immunohistochemistry for proliferation markers (Ki-67, PCNA)

• Cell cycle analysis:

  • Perform flow cytometry with propidium iodide staining to determine cell cycle distribution

  • Compare between NUB1-manipulated and control cells

  • Analyze expression of cell cycle regulators by western blotting

• Apoptosis assays:

  • Flow cytometry using Annexin V/PI staining

  • Overexpression of FLAG-NUB1 in appropriate cell lines (e.g., A498 renal cell carcinoma cells)

  • Assess apoptosis 24 hours post-transfection

Research has shown that NUB1 overexpression can induce apoptosis in up to 90% of A498 renal cell carcinoma cells within 24 hours of transfection .

What are common issues when detecting NUB1 by Western blotting and how can they be resolved?

For western blotting, use reducing conditions and Immunoblot Buffer Group 1 as validated in published protocols .

How should I optimize immunofluorescence staining for NUB1?

For optimal immunofluorescence results with NUB1 antibodies:

• Fixation protocol: Use 4% paraformaldehyde for 15 minutes at room temperature followed by 0.1% Triton X-100 permeabilization for 5 minutes.

• Antibody dilution: Start with 1:200 dilution and optimize based on signal strength. For weak signals, consider using signal amplification systems rather than increasing primary antibody concentration .

• Incubation conditions: Incubate with primary antibody overnight at 4°C to maximize specific binding.

• Positive controls: Include double-immunolabeling with established markers. For example, in neurodegenerative disease research, co-staining with anti-phosphorylated α-synuclein antibodies (1:500) helps validate NUB1 localization in inclusions .

• Counterstaining: Use fluorescein isothiocyanate-conjugated anti-rabbit IgG for NUB1 detection, combined with appropriate secondary antibodies for co-stained proteins (e.g., Texas Red-conjugated anti-mouse IgG) .

What are optimal conditions for inducing and detecting NUB1 expression in cell culture experiments?

NUB1 is an interferon-inducible protein, and its expression can be manipulated under the following conditions:

• IFN-α induction: Treat cells with IFN-α at 1000 IU/ml for 24 hours to induce NUB1 expression. Note that induction patterns vary by cell type; for example, caki-1 and A498 cells show minimal response to IFN-α (1.18-fold and 1.06-fold induction, respectively) .

• Cell line selection: For endogenous NUB1 studies, select responsive cell lines such as 786-0, OCUU3, OS-RC2, OCUU1, ACHN, or 4TUHR, which show significant NUB1 induction following IFN-α treatment .

• Transfection conditions: For overexpression studies, use pcDNA3/FLAG-NUB1 or pcDNA3/FLAG-NUB1L (alternative splicing variant) constructs with standard transfection protocols. Analyze expression 24-48 hours post-transfection .

• RNA interference: For knockdown studies, use validated siRNA sequences targeting nucleotides 458-476 after the NUB1 start codon. Co-transfect siRNA with a marker plasmid (e.g., pNAC-SphI) using Lipofectamine 2000 for optimal results .

How can NUB1 antibodies be used to investigate neurodegenerative diseases?

NUB1 has been implicated in neurodegenerative α-synucleinopathies, including Parkinson's disease. Key experimental approaches include:

• Immunohistochemistry of brain tissues:

  • NUB1 accumulates in inclusion bodies in brain sections from patients with Parkinson's disease and other α-synucleinopathies

  • Use anti-NUB1 antibody (10 μg/ml) alongside anti-phosphorylated α-synuclein antibodies (1:500)

  • Examine sections with fluorescence microscopy (e.g., Olympus Provis fluorescence microscope)

• Quantification of NUB1-positive inclusions:

  • Count the total number of inclusions immunostained with anti-NUB1 and anti-phosphorylated α-synuclein in contiguous sections

  • Compare inclusion formation in different brain regions (brainstem of PD, temporal lobe of DLB, and pons of MSA)

• Co-transfection assays to study inclusion formation:

  • Co-transfect cells with synphilin-1 (a major component of inclusion bodies) and NUB1

  • Assess inclusion formation using immunofluorescence

  • Results indicate that NUB1 suppresses the formation of synphilin-1-positive inclusions

How should I interpret variations in NUB1 expression across different cancer types?

When analyzing NUB1 expression in cancer research:

• Expression level variations: NUB1 shows differential expression across cancer types. For example, NUB1 protein expression is reduced in hepatocellular carcinoma (HCC) tissues and cells compared to normal tissues .

• Correlation with proliferation: Low NUB1 expression correlates with increased cancer cell proliferation in several cancers, as NUB1 normally functions as a negative regulator of cell growth .

• IFN-α sensitivity prediction: In renal cell carcinoma, levels of NUB1 induction by IFN-α can predict tumor responsiveness to IFN-α therapy. Significant correlation exists between NUB1 induction and IFN-α sensitivity in RCC cell lines .

• Relationship with NEDDylation targets:

  • In HCC, reduced NUB1 leads to increased PCNA protein stability by upregulating NEDD8

  • PCNA NEDDylation at lysine 164 antagonizes PCNA K48-linked polyubiquitination

  • This mechanism increases PCNA stability and promotes cancer cell growth

• Therapeutic implications: Targeting the NEDDylation pathway with inhibitors (e.g., TAS4464) can suppress cancer cell growth in NUB1-low tumors by inhibiting PCNA NEDDylation .

What experimental controls are critical when studying NUB1-dependent protein degradation mechanisms?

When investigating NUB1-dependent protein degradation, include these essential controls:

• Proteasome inhibition controls:

  • Include MG132 (15 μM, 12h) treatment groups to verify proteasome-dependent degradation

  • This control is crucial as it should abolish the effects of NUB1 manipulation if the mechanism involves proteasomal degradation

• mRNA expression verification:

  • Perform qRT-PCR to confirm that changes in protein levels are post-transcriptional rather than due to altered gene expression

  • For example, NUB1 knockdown increases PCNA protein but not mRNA levels, confirming post-transcriptional regulation

• Rescue experiments:

  • Perform genetic rescue by re-expressing the target protein

  • For example, NEDD8 knockdown alleviates the increase in PCNA expression caused by NUB1 downregulation

  • Similarly, NEDD8 overexpression attenuates the decrease in PCNA expression caused by NUB1 upregulation

• NEDDylation vs. ubiquitination assessment:

  • Include co-immunoprecipitation controls to differentiate between NEDDylation and ubiquitination effects

  • Compare K48-linked polyubiquitination (degradation signal) with NEDDylation patterns

  • These controls help distinguish competing post-translational modifications that affect protein stability

• Functional validation:

  • Include proliferation or functional assays (EdU, colony formation) to connect molecular changes to cellular phenotypes

  • For example, NEDD8 knockdown alleviates enhanced proliferation induced by NUB1 downregulation