UBAC2 Antibody, HRP conjugated

What is UBAC2 and what cellular functions has it been implicated in?

UBAC2 (Ubiquitin-associated domain-containing protein 2) is a transmembrane protein primarily localized to the endoplasmic reticulum (ER). Recent research has identified UBAC2 as a key player in several critical cellular processes:

• ER-phagy: UBAC2 functions as an ER-phagy receptor that facilitates selective degradation of ER fragments, playing an essential role in maintaining ER homeostasis .

• Cancer biology: UBAC2 has been found to be upregulated in bladder cancer tissues and cell lines, where its knockdown inhibits cancer cell proliferation both in vitro and in vivo .

• Inflammatory regulation: UBAC2 serves as a negative regulator of inflammatory responses, with its deficiency resulting in increased inflammatory activity through disruption of ER homeostasis .

• Cell cycle regulation: Studies have shown that UBAC2 knockdown leads to cell cycle arrest at G0/G1 phase in bladder cancer cell lines .

Research has also identified genetic associations between UBAC2 variants and inflammatory conditions such as Behçet's disease, suggesting its broader role in immune regulation .

How does UBAC2 promote autophagy, particularly ER-phagy?

UBAC2 facilitates ER-phagy through a multi-step mechanism that depends on specific structural domains and protein interactions:

• UBAC2 harbors a canonical LC3-interacting region (LIR) motif (specifically WNRL) in its cytoplasmic domain, which enables direct binding to autophagosomal proteins, particularly GABARAP .

• Upon cellular stress conditions (such as starvation or ER stress), UBAC2 undergoes phosphorylation at serine 223 by microtubule affinity-regulating kinase 2 (MARK2) .

• This phosphorylation promotes UBAC2 dimerization, which significantly enhances its binding affinity for GABARAP .

• The strengthened interaction between dimerized UBAC2 and GABARAP facilitates the selective targeting and degradation of ER fragments through autophagy .

Experimental evidence has demonstrated that UBAC2 knockout significantly reduces ER-phagy flux under both starvation-induced autophagy and ER-stress conditions in multiple cell lines, including HeLa, THP-1, and HT-29 cells .

What are the optimal sample preparation methods for UBAC2 detection using antibodies?

For optimal UBAC2 detection using antibodies, researchers should follow these validated protocols based on published research:

For protein extraction and western blotting:

• Extract total protein using RIPA lysis buffer (Invitrogen or equivalent).

• Determine protein concentration using BCA Protein Assay Kit.

• Separate proteins on 10% SDS-PAGE gels.

• Transfer proteins to polyvinylidene difluoride membranes.

• Block membranes for 1 hour at room temperature.

• Probe with primary UBAC2 antibody (such as Cat No 25122-1-AP from Proteintech) overnight at 4°C.

• Incubate with HRP-conjugated secondary antibodies for 1 hour at room temperature.

• Develop images using ECL kit and appropriate imaging system .

For immunohistochemistry:
Immunohistochemistry analysis has been successfully employed to compare UBAC2 protein expression between bladder cancer tissues and adjacent normal tissues, confirming significantly higher expression in cancer tissues .

For immunofluorescence:
Immunofluorescence analysis has revealed that UBAC2 is primarily localized in the cytoplasm of bladder cancer cells . When studying UBAC2 co-localization with autophagy proteins like GABARAP, confocal microscopy with appropriate fluorescent-labeled antibodies is recommended .

How is UBAC2 involved in cancer progression and what are the best experimental approaches to study this role?

UBAC2 demonstrates significant oncogenic potential in bladder cancer through several mechanisms:

Expression and prognostic value:

• UBAC2 mRNA and protein levels are significantly upregulated in bladder cancer tissues compared to surrounding normal tissues .

• Higher UBAC2 expression correlates with poorer survival rates in bladder cancer patients, as demonstrated by Kaplan-Meier survival analyses of both hospital cohorts (48 cases) and TCGA database (406 cases) .

Molecular mechanisms:

• UBAC2 promotes cancer cell proliferation by affecting cell cycle regulation, specifically by suppressing p27 expression .

• UBAC2 interacts with circular RNA BCRC-3, affecting its interaction with miR-182-5p, which ultimately impacts p27 expression and cell cycle progression .

Recommended experimental approaches:

• RNA interference studies: Using shRNA targeting UBAC2 in cancer cell lines to assess effects on proliferation, cell cycle, and expression of key proteins like p27.

• RNA immunoprecipitation (RIP): To investigate interactions between UBAC2 and circular RNAs such as BCRC-3.

• Luciferase reporter assays: To examine the effect of UBAC2 on p27 3′-UTR activity.

• Xenograft models: To verify findings in vivo, as knockdown of UBAC2 has been shown to significantly inhibit tumor growth in nude mice .

What is the significance of UBAC2 phosphorylation and dimerization in experimental design?

UBAC2 undergoes critical post-translational modifications that significantly alter its function, particularly in the context of ER-phagy:

Phosphorylation mechanism:

• Under ER stress or autophagy activation, microtubule affinity-regulating kinase 2 (MARK2) phosphorylates UBAC2 at serine 223 .

• This phosphorylation event serves as a molecular switch that promotes UBAC2 dimerization .

Functional consequences:

• Dimerized UBAC2 demonstrates significantly enhanced binding affinity for GABARAP compared to monomeric UBAC2 .

• This strengthened interaction accelerates ER-phagy progression, increasing the selective degradation of ER fragments .

Experimental design considerations:

• Phosphorylation site mutants: Generate S223A mutants of UBAC2 to prevent phosphorylation and assess functional outcomes.

• Dimerization assays: Employ methods to quantify UBAC2 dimerization under different cellular stress conditions.

• Co-immunoprecipitation studies: Compare GABARAP binding between wild-type UBAC2 and phosphorylation-deficient mutants.

• Kinase inhibition: Use specific MARK2 inhibitors to assess their impact on UBAC2 phosphorylation, dimerization, and subsequent ER-phagy efficiency.

Understanding these modifications is crucial for designing experiments that accurately capture UBAC2 function in different cellular contexts and stress conditions.

How do mutations in the LIR motif of UBAC2 affect its function and detection?

The LC3-interacting region (LIR) motif of UBAC2 is essential for its autophagy-related functions. Mutations in this region have significant implications for both function and detection:

LIR motif characteristics:

• UBAC2 contains a highly conserved LIR motif (WNRL) that mediates direct binding to ATG8-family proteins, particularly GABARAP .

• This interaction is crucial for UBAC2's function as an ER-phagy receptor .

Impact of LIR mutations:

• Functional impairment: UBAC2 LIR mutants (W275A and L278A) fail to interact with GABARAP, abolishing co-localization and significantly reducing ER-phagy flux .

• Inflammatory consequences: LIR motif mutations decrease ER-phagy efficiency and increase sterile inflammation associated with ER stress in vivo, making experimental animals more susceptible to dextran sulfate sodium (DSS)-induced ulcerative colitis .

Detection considerations:

• Antibodies targeting regions that include or are affected by the LIR motif might show altered binding patterns in LIR-mutant variants.

• When studying the interaction between UBAC2 and ATG8 family proteins, LIR mutants serve as important negative controls to validate antibody specificity.

Experimental approach recommendation:
Generate and compare wild-type UBAC2 and LIR-mutant (W275A and L278A) constructs in functional assays to determine the specific contribution of the LIR motif to the biological process under investigation.

What are the optimal conditions for using HRP-conjugated UBAC2 antibodies in western blotting?

Based on published research methodologies for UBAC2 detection, the following optimized western blotting protocol is recommended:

Sample preparation:

• Extract total protein using RIPA lysis buffer (Invitrogen or equivalent commercial product).

• Determine protein concentration using BCA Protein Assay Kit (Beyotime or similar).

• Prepare protein samples with loading buffer and denature at 95°C for 5 minutes.

Gel electrophoresis and transfer:

• Separate 20-50 μg of protein on 10% SDS-PAGE gels.

• Transfer proteins to polyvinylidene difluoride (PVDF) membranes using standard transfer conditions (typically 100V for 90 minutes or 20V overnight at 4°C).

Antibody incubation:

• Block membranes with 5% non-fat milk in TBST for 1 hour at room temperature.

• Incubate with primary UBAC2 antibody (such as Cat No 25122-1-AP from Proteintech) at a 1:1000 dilution overnight at 4°C.

• Wash membranes 3-5 times with TBST, 5 minutes each.

For direct HRP-conjugated UBAC2 antibodies:

• Skip secondary antibody step and proceed directly to detection after washing.

• Optimize concentration according to manufacturer's recommendations, typically 1:500 to 1:2000 dilution.

For unconjugated primary antibodies:

• Incubate with HRP-conjugated secondary antibody at 1:5000 dilution for 1 hour at room temperature.

• Wash membranes 3-5 times with TBST, 5 minutes each.

Detection:

• Develop images using ECL kit (such as those from Servicebio) according to manufacturer's instructions.

• Capture images using a chemiluminescence imaging system (such as BioSpectrum600) .

Troubleshooting tips:

• If background is high, increase washing duration or frequency, or reduce antibody concentration.

• If signal is weak, increase protein loading, antibody concentration, or exposure time.

• Include appropriate positive controls (bladder cancer cell lines such as RT4, EJ, UMUC3, T24, and T24T are known to express higher levels of UBAC2 compared to normal cells) .

How can RNA immunoprecipitation (RIP) be performed with UBAC2 antibodies?

RNA immunoprecipitation (RIP) is a valuable technique for studying RNA-protein interactions involving UBAC2. Based on published protocols, the following methodology is recommended:

Protocol overview:

• Harvest approximately 1.5 × 10^7 cells and lyse to extract total protein.

• Immunoprecipitate total protein with antibody against UBAC2 (such as Cat. No. 25122-1-AP from Proteintech Group) or IgG antibody as control.

• Use Protein A/G magnetic beads (Life Technologies) for precipitation.

• Purify RNA complexes combining on the protein with RNeasy Mini Kit (QIAGEN).

• Analyze by RT-PCR for RNA of interest .

Critical considerations for successful RIP:

• Cross-linking: Consider whether formaldehyde cross-linking is necessary based on the strength of the RNA-protein interaction.

• RNase inhibitors: Include RNase inhibitors in all buffers to prevent RNA degradation.

• Washing stringency: Optimize washing conditions to minimize background while maintaining specific interactions.

• Controls: Always include IgG antibody control to account for non-specific binding.

• Validation: Confirm successful immunoprecipitation of UBAC2 by western blot before proceeding to RNA analysis.

This technique has been successfully applied to demonstrate the interaction between UBAC2 and circular RNA BCRC-3, which affects miR-182-5p binding and subsequently regulates p27 expression .

What methods can be used to study UBAC2 interactions with autophagy-related proteins?

Multiple complementary approaches can be employed to study UBAC2 interactions with autophagy-related proteins, particularly with ATG8-family proteins like GABARAP:

Co-immunoprecipitation (Co-IP):

• Prepare cell lysates under native conditions to preserve protein-protein interactions.

• Immunoprecipitate with anti-UBAC2 antibodies (or anti-GABARAP antibodies for reverse Co-IP).

• Analyze precipitated complexes by western blotting for interacting partners.

This approach has successfully demonstrated that UBAC2 interacts with LC3 (MAP1LC3A/B/C), GABARAP, and GABARAPL2, with the strongest association observed between UBAC2 and GABARAP .

Proximity ligation assay (PLA):
This technique can visualize protein-protein interactions in situ with high sensitivity and specificity, providing spatial information about UBAC2 interactions.

Confocal microscopy for co-localization:

• Perform immunofluorescence staining with antibodies against UBAC2 and potential interacting partners.

• Analyze co-localization using confocal microscopy.

Confocal analysis has confirmed that UBAC2 colocalizes with GABARAP during autophagy activation .

Mutation analysis:
Generate mutants of key domains (such as the LIR motif) and assess their impact on protein interactions. For example:

• UBAC2 LIR mutant (W275A and L278A) fails to interact with GABARAP, confirming the importance of this motif for the interaction .

Quantitative measurements:
The association between UBAC2 and GABARAP significantly increases upon treatment with thapsigargin (TG) or EBSS medium (starvation conditions), highlighting the dynamic nature of these interactions under different cellular conditions .

How can the specificity of UBAC2 antibodies be validated for research applications?

Validating antibody specificity is crucial for reliable research outcomes. For UBAC2 antibodies, the following validation approaches are recommended:

Genetic knockout/knockdown controls:

• Generate UBAC2 knockout cell lines using CRISPR-Cas9 or stable knockdown cells using shRNA (shUBAC2).

• Compare antibody signals between wild-type and knockout/knockdown samples using western blot and immunostaining.

This approach has been successfully employed in published research, where four shRNAs targeting the coding region of UBAC2 were designed and stably transfected into EJ and UMUC3 cells, with knockdown efficiency verified by qRT-PCR and western blotting .

Overexpression controls:

• Transfect cells with UBAC2 expression constructs (wild-type or tagged versions).

• Confirm increased antibody signal in overexpressing cells.

Cross-reactivity assessment:
Test antibody on samples from multiple species if studying UBAC2 across different model organisms.

Multiple antibody validation:
Use antibodies from different sources or targeting different epitopes to confirm consistent results.

Antibody blocking:
Preincubate antibody with purified UBAC2 protein or peptide and confirm signal reduction.

Correlation with mRNA levels:
Compare protein detection with mRNA levels as measured by qRT-PCR for concordance.

Specificity in diverse applications:
Validate antibody performance in each specific application context (western blot, immunoprecipitation, immunohistochemistry, etc.), as performance may vary across applications.

How can UBAC2 antibodies be used to investigate inflammatory conditions?

UBAC2 has been identified as a negative regulator of inflammatory responses, with significant implications for inflammatory conditions:

Role in inflammatory regulation:

• UBAC2 restrains inflammatory responses through its function as an ER-phagy receptor .

• UBAC2 deficiency results in inflammatory responses through disruption of ER homeostasis .

• UBAC2 variants from inflammatory diseases or mutations in the LIR motif decrease ER-phagy flux and increase sterile inflammation associated with ER stress in vivo .

Experimental approaches:

• Tissue expression studies: Use UBAC2 antibodies to compare expression levels in inflamed versus normal tissues from patient samples or animal models.

• Genetic association: Study the connection between UBAC2 genetic variants and inflammatory diseases, as demonstrated in Behçet's disease research .

• Animal models: UBAC2-deficient mice are more susceptible to dextran sulfate sodium (DSS)-induced ulcerative colitis, providing a model system to study UBAC2's role in inflammation .

• Cell signaling analysis: Investigate how UBAC2 expression affects inflammatory signaling pathways using UBAC2 antibodies in combination with phospho-specific antibodies for key inflammatory mediators.

Clinical relevance:

• The MARK2-UBAC2 axis that regulates ER-phagy may provide targets for the treatment of inflammatory diseases .

• Monitoring UBAC2 expression or activation status could potentially serve as a biomarker for inflammatory conditions or treatment response.

What experimental designs can elucidate the dual roles of UBAC2 in cancer and autophagy?

UBAC2 functions in both cancer promotion and autophagy regulation suggest complex cellular roles that require sophisticated experimental designs to elucidate:

Integrated experimental approaches:

• Conditional knockout systems:

  • Generate cell lines with inducible UBAC2 knockout/knockdown

  • Compare effects on both cancer hallmarks (proliferation, migration) and autophagy markers

  • Use systems like Tet-On/Tet-Off to regulate UBAC2 expression temporally

• Domain-specific mutants:

  • Create UBAC2 constructs with mutations in specific functional domains

  • LIR motif mutants (W275A and L278A) to disrupt autophagy functions

  • Phosphorylation site mutants (S223A) to prevent dimerization

  • Test each mutant's effect on both cancer progression and autophagy

• Dual reporter systems:

  • Combine proliferation markers with autophagy flux reporters

  • Monitor both processes simultaneously in response to UBAC2 modulation

• Context-dependent analysis:

  • Compare UBAC2 functions under different stress conditions (nutrient deprivation, ER stress, hypoxia)

  • Determine if cancer promotion and autophagy regulation are linked or independent functions

• Interactome mapping:

  • Use proteomics approaches to identify UBAC2 binding partners

  • Classify partners into cancer-related versus autophagy-related proteins

  • Validate key interactions using co-immunoprecipitation with UBAC2 antibodies

This comprehensive approach can help determine whether UBAC2's roles in cancer and autophagy represent separate functions or integrated cellular mechanisms that contribute to disease progression.

What are common challenges with UBAC2 antibodies and their solutions?

When working with UBAC2 antibodies, researchers may encounter several challenges that can affect experimental outcomes:

Challenge 1: Weak or inconsistent signals

• Possible causes: Low UBAC2 expression in sample, antibody degradation, inefficient protein extraction

• Solutions:

  • Verify UBAC2 expression levels in your experimental system (RT4, EJ, UMUC3, T24, and T24T bladder cancer cell lines have confirmed high UBAC2 expression)

  • Use fresh antibody aliquots and avoid repeated freeze-thaw cycles

  • Optimize protein extraction using RIPA buffer with protease and phosphatase inhibitors

  • Increase antibody concentration or incubation time

Challenge 2: High background

• Possible causes: Non-specific binding, insufficient blocking, concentrated antibody

• Solutions:

  • Increase blocking time (at least 1 hour at room temperature)

  • Optimize antibody dilution through titration experiments

  • Include additional washing steps between antibody incubations

  • Use more stringent washing buffers

Challenge 3: Inconsistent results in phosphorylation studies

• Possible causes: Rapid phosphorylation dynamics, phosphatase activity during sample preparation

• Solutions:

  • Include phosphatase inhibitors in all buffers

  • Standardize time between stimulation and sample collection

  • Consider using phosphorylation-specific antibodies if available

  • Include positive controls treated with phosphatase inhibitors

Challenge 4: Discrepancies between protein and mRNA levels

• Possible causes: Post-transcriptional regulation, protein stability differences

• Solutions:

  • Compare results with both qRT-PCR and protein detection methods

  • Consider protein half-life studies with cycloheximide chase

  • Analyze samples at multiple time points to capture dynamics

Challenge 5: Variable performance across applications

• Possible causes: Different epitope accessibility in different applications

• Solutions:

  • Validate antibody performance specifically for each application

  • Consider using different antibodies optimized for specific applications

  • Use appropriate positive and negative controls for each application

How can contradictory data from UBAC2 studies be reconciled and interpreted?

When encountering contradictory results in UBAC2 research, several analytical approaches can help reconcile disparate findings:

1. Context-dependent functions analysis:

• UBAC2 functions may vary significantly based on cellular context

• Compare experimental conditions between contradictory studies:

  • Cell types used (cancer vs. normal; tissue origin)

  • Stress conditions (basal, starvation, ER stress)

  • Disease models (cancer, inflammation)

2. Isoform-specific effects:

• Consider whether different UBAC2 splice variants might explain contradictory results

• The study of rs7999348 in Behçet's disease showed this SNP tags a functional variant associated with increased mRNA expression of a specific UBAC2 transcript variant

• Verify which isoforms are expressed in your experimental system

3. Post-translational modification status:

• UBAC2 phosphorylation at S223 by MARK2 significantly affects its function

• Inconsistent results may stem from different phosphorylation states

• Consider analyzing phosphorylation status alongside total protein levels

4. Methodological reconciliation:

• Create a comprehensive experimental approach that tests contradictory findings under identical conditions

• Include appropriate controls for each variable that might explain discrepancies

• Consider time-course experiments to capture dynamic changes that might explain seemingly contradictory snapshot results

5. Data integration framework:

• When interpreting contradictory data, construct a model that accommodates seemingly contradictory results as context-dependent functions

• Test model predictions with new experiments designed to directly address contradictions

• Consider mathematical modeling to integrate complex datasets and identify parameters that might explain variability

By systematically analyzing contradictory findings through these approaches, researchers can develop more comprehensive models of UBAC2 function that account for its diverse roles in different cellular contexts.