UBXN8 Antibody

What is UBXN8 and what are its primary cellular functions?

UBXN8, also known as UBXD6 or REP-8, is a member of the ubiquitin regulatory X (UBX) protein family, which represents the largest known group of p97 cofactors. It functions as a transmembrane protein localized in the endoplasmic reticulum (ER) membrane. UBXN8 tethers p97, a versatile ATPase complex, to the ER membrane via its UBX domain. This association facilitates the efficient ER-associated degradation (ERAD) of misfolded proteins .

Research indicates UBXN8 may have multiple cellular functions:

• Involvement in protein quality control through ERAD mechanisms

• Potential tumor suppressor activity

• Possible role in reproductive processes

Recent findings suggest UBXN8 may function as a tumor suppressor in a TP53-dependent manner in hepatocellular carcinoma, and its silencing appears to contribute to leukemogenesis in t(8;21) acute myeloid leukemia .

What types of UBXN8 antibodies are available for research applications?

Several types of UBXN8 antibodies are available for research purposes, varying in species reactivity, conjugation, and applications:

The primary commercially available UBXN8 antibody is a polyclonal rabbit antibody directed towards the middle region of human UBXN8, with the immunogen being a synthetic peptide with the sequence: RKLEERFYQMTGEAWKLSSGHKLGGDEGTSQTSFETSNREAAKSQNLPKP .

How should UBXN8 antibodies be stored and handled to maintain optimal activity?

For optimal preservation of UBXN8 antibody activity, researchers should follow these evidence-based storage and handling protocols:

• Store antibodies at 4°C for short-term use (typically up to one week)

• For long-term storage, aliquot and maintain at -20°C

• Avoid repeated freeze-thaw cycles which can degrade antibody performance

• Antibodies are typically shipped with polar packs and should be stored immediately upon receipt at the recommended temperature

• Most UBXN8 antibodies are formulated in PBS with 2% sucrose and 0.09% sodium azide as a preservative

• Antibody concentration is typically 0.5 mg/ml

Following these practices will help maintain antibody specificity and sensitivity for experimental applications.

How is UBXN8 implicated in cancer biology, particularly in acute myeloid leukemia?

Recent research has revealed critical roles for UBXN8 in cancer biology, particularly in t(8;21) acute myeloid leukemia (AML). The RUNX1-RUNX1T1 fusion protein, resulting from the t(8;21) chromosomal translocation, triggers heterochromatic silencing of the UBXN8 gene through specific epigenetic mechanisms .

The mechanism involves:

• RUNX1-RUNX1T1 binding to RUNX1-binding sites in the UBXN8 promoter region

• Recruitment of chromatin-remodeling enzymes including DNMTs (DNA methyltransferases)

• Methylation of CpG islands in the UBXN8 promoter

• Epigenetic silencing of UBXN8 expression

Research demonstrates that restoring UBXN8 expression can:

• Inhibit proliferation and colony-forming ability of t(8;21) AML cell lines

• Promote cell cycle arrest in G1 phase

• Significantly slow tumor proliferation in vivo

• Promote differentiation of RUNX1-RUNX1T1-positive cells

These findings suggest UBXN8 functions as a tumor suppressor in AML, and enhancing its expression may represent a potential therapeutic approach for t(8;21) AML patients.

What experimental methods are most effective for studying UBXN8 expression in leukemia models?

For investigating UBXN8 expression in leukemia models, researchers have successfully employed several complementary methodologies:

• RNA Expression Analysis:

  • qRT-PCR using SYBR Green with GAPDH as internal control

  • Analysis using the 2^(-ΔΔCt) method for relative quantification

• Protein Expression Analysis:

  • Western blot using anti-UBXN8 antibodies (such as Abcam ab159924)

  • β-actin (Abcam ab179467) as loading control

• Epigenetic Regulation Studies:

  • Chromatin Immunoprecipitation (ChIP) using antibodies against:

    • RUNX1 (Abcam ab23980)

    • RUNX1T1 (Abcam ab195329)

    • DNMTs (DNMT3A, DNMT3B, DNMT1)

  • MethylC-capture sequencing (MCC-Seq) to detect methylation patterns

• Functional Studies:

  • Lentiviral vectors for UBXN8 overexpression

  • Decitabine treatment (1.0 μM for 72h) to restore UBXN8 expression through DNA demethylation

These approaches provide comprehensive insights into UBXN8 regulation, expression, and function in leukemia models.

What are the challenges in developing UBXN8-targeted therapies and how might they be overcome?

Developing UBXN8-targeted therapies presents several significant challenges that researchers are working to address:

• Selective Epigenetic Modulation:

  • Challenge: Broadly acting DNA methyltransferase inhibitors like decitabine can restore UBXN8 expression but affect numerous genes

  • Solution: Development of locus-specific epigenetic editors using CRISPR-based approaches to selectively demethylate the UBXN8 promoter

• Tissue-Specific Delivery:

  • Challenge: Delivering UBXN8-enhancing treatments specifically to leukemic cells

  • Solution: Nanoparticle-based delivery systems or targeted vectors that preferentially affect cells expressing RUNX1-RUNX1T1

• Combination Therapy Optimization:

  • Challenge: Determining optimal combinations with conventional chemotherapy

  • Solution: Systematic testing of UBXN8-enhancing agents with different chemotherapeutic regimens in preclinical models

• Resistance Mechanisms:

  • Challenge: Cancer cells may develop alternative pathways to bypass UBXN8 tumor-suppressive effects

  • Solution: Identifying and targeting parallel pathways simultaneously

• Biomarker Development:

  • Challenge: Identifying patients most likely to benefit from UBXN8-targeted therapies

  • Solution: Developing assays to measure UBXN8 promoter methylation status as predictive biomarkers

What controls should be included when performing Western blot analysis with UBXN8 antibodies?

When conducting Western blot analysis with UBXN8 antibodies, researchers should implement a comprehensive panel of controls to ensure result validity:

Essential Controls:

• Positive Control:

  • HCT15 whole cell lysates have been validated for UBXN8 detection

  • Known UBXN8-expressing cell lines (SKNO-1, Kasumi-1, U-937) for leukemia research

• Negative Control:

  • Cell lines with confirmed low UBXN8 expression (SKNO-siAE cells with RUNX1-RUNX1T1 knockdown)

  • Primary antibody omission to assess secondary antibody specificity

• Loading Control:

  • β-actin (using antibodies such as Abcam ab179467)

  • GAPDH or other housekeeping proteins to normalize protein loading

• Antibody Specificity Controls:

  • Peptide competition assay using the immunizing peptide (RKLEERFYQMTGEAWKLSSGHKLGGDEGTSQTSFETSNREAAKSQNLPKP)

  • Use of multiple UBXN8 antibodies targeting different epitopes for verification

• Sample Preparation Controls:

  • Inclusion of protease inhibitors in lysis buffers to prevent degradation

  • Denaturation temperature and time optimization for the 30 kDa UBXN8 protein

Recommended antibody dilution for Western blot is 1.0 μg/ml, though optimization may be required for different experimental systems .

How can researchers effectively modulate UBXN8 expression in experimental models?

Researchers have successfully employed several strategies to modulate UBXN8 expression in experimental models:

Upregulation Methods:

• Lentiviral Expression Systems:

  • Commercially available lentiviral vectors (e.g., from GeneChem) for stable UBXN8 overexpression

  • Protocol: Transduction at MOI of 100, centrifugation at 1000 × g for 3h with 5 ng/mL polybrene, followed by puromycin selection for 48h

  • Selection of stable clones over 3 weeks with confirmation by flow cytometry

• Epigenetic Modulation:

  • Decitabine treatment (1.0 μM for 72h) effectively upregulates UBXN8 expression in RUNX1-RUNX1T1+ cell lines

  • Other DNMT inhibitors like 5-azacytidine may also restore expression

Downregulation Methods:

• RNA Interference:

  • siRNA targeting UBXN8 mRNA

  • shRNA for stable knockdown in long-term experiments

• CRISPR/Cas9 Gene Editing:

  • Generation of UBXN8 knockout cell lines for loss-of-function studies

  • CRISPR interference (CRISPRi) for reversible gene silencing

Detection and Verification:

• qRT-PCR for mRNA expression analysis

• Western blot for protein expression confirmation

• Functional assays to validate biological effects

These approaches provide researchers with a toolkit for manipulating UBXN8 expression to study its functions in various experimental contexts.

What are the optimal protocols for immunohistochemical detection of UBXN8 in tissue samples?

For optimal immunohistochemical detection of UBXN8 in tissue samples, researchers should follow this evidence-based protocol:

Sample Preparation:

• Fix tissue samples in 10% neutral buffered formalin for 24-48 hours

• Process and embed in paraffin

• Section tissues at 4-5 μm thickness

• Mount on positively charged slides

Antigen Retrieval:

• Deparaffinize sections in xylene and rehydrate through graded alcohols

• Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) for 20 minutes

• Cool slides to room temperature for 20 minutes

• Wash in PBS (3 × 5 minutes)

Staining Procedure:

• Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

• Protein block with 5% normal goat serum for 30 minutes

• Incubate with primary UBXN8 antibody (1:100-1:200 dilution) at 4°C overnight

• Wash in PBS (3 × 5 minutes)

• Apply HRP-conjugated secondary antibody for 30 minutes at room temperature

• Wash in PBS (3 × 5 minutes)

• Develop with DAB substrate

• Counterstain with hematoxylin

• Dehydrate, clear, and mount with permanent mounting medium

Controls and Validation:

• Positive control: Include known UBXN8-expressing tissues

• Negative controls:

  • Primary antibody omission

  • Non-specific IgG substitution

  • UBXN8-negative tissues

Analysis Considerations:

• Assess both staining intensity and percentage of positive cells

• Document subcellular localization (membrane/cytoplasmic/nuclear)

• Consider dual immunofluorescence with ER markers to confirm localization

What are common problems encountered when using UBXN8 antibodies and how can they be resolved?

Researchers commonly encounter several issues when working with UBXN8 antibodies. The following troubleshooting guide addresses these challenges:

Problem 1: Weak or No Signal in Western Blot

• Potential Causes:

  • Insufficient antibody concentration

  • Inadequate antigen

  • Suboptimal transfer

  • Protein degradation

• Solutions:

  • Increase antibody concentration (recommended starting concentration: 1.0 μg/ml)

  • Increase protein loading (30-50 μg total protein)

  • Optimize transfer conditions for the 30 kDa UBXN8 protein

  • Add fresh protease inhibitors to lysis buffer

  • Ensure samples are kept cold during preparation

Problem 2: High Background in Immunostaining

• Potential Causes:

  • Insufficient blocking

  • Excessive antibody concentration

  • Cross-reactivity

  • Inadequate washing

• Solutions:

  • Extend blocking time with 5% BSA or normal serum

  • Titrate antibody to optimal concentration

  • Pre-absorb antibody with the immunizing peptide

  • Increase washing duration and frequency

Problem 3: Inconsistent Results Between Experiments

• Potential Causes:

  • Antibody degradation

  • Variable UBXN8 expression

  • Protocol inconsistencies

• Solutions:

  • Aliquot antibodies to avoid freeze-thaw cycles

  • Standardize cell culture conditions that may affect UBXN8 expression

  • Develop detailed protocols with timing specifications

  • Include consistent positive controls across experiments

Problem 4: Unexpected Molecular Weight Bands

• Potential Causes:

  • Post-translational modifications

  • Alternative splice variants

  • Non-specific binding

• Solutions:

  • Compare with recombinant UBXN8 protein as positive control

  • Validate with knockout or knockdown controls

  • Test multiple antibodies targeting different epitopes

How can researchers quantify UBXN8 expression levels accurately across different experimental conditions?

For accurate quantification of UBXN8 expression across experimental conditions, researchers should implement these methodological approaches:

mRNA Quantification:

• qRT-PCR Optimization:

  • Design primers spanning exon-exon junctions to avoid genomic DNA amplification

  • Validate primer efficiency using standard curves

  • Use multiple reference genes (GAPDH, β-actin, 18S rRNA) for normalization

  • Calculate relative expression using the 2^(-ΔΔCt) method

Protein Quantification:

• Western Blot Densitometry:

  • Include serial dilutions of samples to ensure detection within linear range

  • Use recombinant UBXN8 standards for absolute quantification

  • Normalize to loading controls (β-actin, GAPDH)

  • Employ image analysis software with background subtraction

  • Perform technical triplicates for statistical validity

• ELISA Development:

  • Sandwich ELISA using capture and detection antibodies against different UBXN8 epitopes

  • Standard curves using recombinant UBXN8 protein

  • Include spike-in controls to assess recovery

Single-Cell Analysis:

• Flow Cytometry:

  • Optimize fixation and permeabilization for intracellular staining

  • Use FITC-conjugated UBXN8 antibodies for direct detection

  • Include fluorescence-minus-one (FMO) controls

  • Analyze mean fluorescence intensity (MFI) for quantification

Data Integration:

• Correlate mRNA and protein expression data

• Account for post-transcriptional regulation

• Normalize to cell number or tissue weight for cross-sample comparison

What considerations should be made when studying UBXN8 in different species or cell types?

When investigating UBXN8 across different species or cell types, researchers should address several important considerations:

Species-Specific Variations:

• Sequence Homology Analysis:

  • Human UBXN8 shares variable homology with orthologs in other species

  • Confirm antibody epitope conservation across species before application

  • Consider species-specific antibodies when available (human, mouse, bovine)

• Expression Pattern Differences:

  • UBXN8 expression levels vary significantly between tissues and species

  • Baseline expression should be established for each model organism

  • Use species-appropriate positive controls

Cell Type Considerations:

• Baseline Expression Mapping:

  • Document normal UBXN8 expression across cell lineages

  • Hematopoietic cells show variable expression with RUNX1-RUNX1T1 status being a significant determinant in AML cells

  • Establish reference expression panels for accurate comparative studies

• Subcellular Localization:

  • UBXN8 predominantly localizes to the ER membrane

  • Confirm localization in each cell type with co-localization studies

  • Consider cell type-specific interaction partners

Methodological Adaptations:

• Extraction Protocols:

  • Optimize lysis conditions for different tissues (harder tissues may require stronger detergents)

  • Adjust membrane protein extraction protocols for tissue-specific lipid compositions

  • Consider subcellular fractionation to enrich ER-associated UBXN8

• Antibody Selection:

  • Choose antibodies validated for your species of interest

  • Available options include:

    • Human-reactive: Multiple formats (unconjugated, HRP, FITC, biotin)

    • Mouse-reactive: Recombinant proteins available

    • Bovine-reactive: Recombinant proteins available

How might UBXN8 function in the context of ER stress and the unfolded protein response?

UBXN8's role in ER stress and the unfolded protein response (UPR) represents an emerging area of investigation with significant implications:

Molecular Mechanisms:

• ERAD Pathway Integration:

  • UBXN8 tethers the p97 ATPase complex to the ER membrane via its UBX domain

  • This facilitates the extraction and degradation of misfolded proteins from the ER

  • Under ER stress conditions, UBXN8 may represent a rate-limiting factor in ERAD efficiency

• Stress Sensor Interactions:

  • Potential interactions with canonical UPR sensors (IRE1α, PERK, ATF6)

  • May modulate the threshold for UPR activation through regulation of misfolded protein load

  • Could influence the switch between adaptive and terminal UPR outcomes

Pathological Implications:

• Cancer Context:

  • Cancer cells often exhibit chronic ER stress due to high protein synthesis rates

  • Downregulation of UBXN8 in AML potentially compromises ERAD efficiency

  • This may create a permissive environment for oncogenic signaling despite elevated ER stress

  • Targeting this vulnerability could selectively affect cancer cells with dysregulated UBXN8

• Neurodegenerative Disease Relevance:

  • Protein misfolding is central to many neurodegenerative conditions

  • UBXN8's role in ERAD may influence disease progression

  • Potential therapeutic target for enhancing proteostasis in neurodegeneration

Research Methodologies:

• Chemical induction of ER stress (tunicamycin, thapsigargin) in UBXN8-modulated cells

• Analysis of UPR markers (XBP1 splicing, ATF4/CHOP induction) with altered UBXN8 expression

• Proteomics to identify UBXN8-dependent ERAD substrates under stress conditions

What is the significance of UBXN8 in tumor suppression beyond AML, and how can this be investigated?

The tumor suppressor potential of UBXN8 extends beyond AML, with emerging evidence for its role in multiple cancer types:

Current Evidence Base:

• Hepatocellular Carcinoma:

  • UBXN8 identified as a tumor suppressor candidate in HCC

  • Functions in a TP53-dependent manner

  • Downregulation associated with poor prognosis

• AML Model:

  • Epigenetically silenced by RUNX1-RUNX1T1 fusion protein

  • Restoration inhibits proliferation and promotes differentiation

  • Overexpression reduces colony formation capacity

Research Approaches for Other Cancer Types:

• Pan-Cancer Expression Analysis:

  • Mining TCGA and other cancer genomics databases for UBXN8 expression patterns

  • Correlation with clinicopathological features and survival outcomes

  • Analysis of promoter methylation status across cancer types

• Mechanistic Investigations:

  • Determine if UBXN8 regulates critical oncoproteins through ERAD

  • Identify cancer-specific binding partners through proteomics

  • Investigate regulation of cell cycle checkpoints in various cancer models

• In Vivo Validation:

  • Generate conditional UBXN8 knockout mouse models for tissue-specific cancer studies

  • Xenograft models with UBXN8 modulation in different cancer cell lines

  • Response to standard therapies with altered UBXN8 expression

Potential Therapeutic Implications:

• Development of specific UBXN8 demethylating agents

• Combination strategies with existing therapies

• Biomarker development for patient stratification

This emerging area of research suggests UBXN8 may have broader tumor suppressor functions across multiple cancer types, potentially through its role in protein quality control and cellular homeostasis.