URI1 Antibody

What is URI1 and what are its primary cellular functions?

URI1 (unconventional prefoldin RPB5 interactor) is a member of the prefoldin family of molecular chaperones that functions as a scaffolding protein involved in ubiquitination and transcription. It interacts with RNA polymerase II subunit RPB5, playing critical roles in cellular regulation. URI1 has multiple subcellular localizations, including the nucleus, cytoplasm, and mitochondria. It specifically colocalizes with several proteins including PFDN2, PFDN4, PPP1CC, RPS6KB1, and STAP1 at mitochondria . URI1 is encoded by a gene originally identified as C19orf2 (Chromosome 19 open reading frame 2) and is also known by several synonyms including RMP (RPB5 mediating protein) and PPP1R19 (Protein phosphatase 1 regulatory subunit 19) .

What is the clinical significance of URI1 amplification in cancer?

URI1 amplification has significant clinical implications in multiple cancer types. In uterine carcinosarcoma (UCS), URI1 amplification was detected in 40% of primary cases compared to only 5.5% in uterine corpus endometrial carcinomas . This has profound prognostic significance: UCS patients with URI1 amplification exhibited only 13% tumor-free survival compared to 41% in patients without URI1 amplification (P=0.023) . Multivariate analysis identified URI1 amplification (OR=6.54, P=0.027) as an independent predictor of poor survival . Additionally, patients with URI1 amplification showed decreased transcription of tumor suppressor and apoptotic regulator genes while exhibiting increased expression of genes regulating oncogenesis, survival, and metastasis . URI1 amplification has also been reported in approximately 10% of ovarian cancers, where increased URI protein levels correlate with tumor aggressiveness .

What are the key specifications researchers should know when selecting a URI1 antibody?

When selecting a URI1 antibody, researchers should consider several critical specifications:

Researchers should note that the observed molecular weight on Western blots may not always match the calculated 60 kDa due to post-translational modifications or alternative splicing. Multiple bands may be detected if a protein sample contains different modified forms simultaneously .

What validated applications exist for URI1 antibodies in research?

URI1 antibodies have been validated for several key research applications:

• Western Blotting: URI1 antibodies have been verified in Western blotting using 293T cell samples, with recommended dilutions ranging from 1:500 to 1:2000 . These applications are critical for detecting URI1 protein levels in experimental manipulations such as URI1 knockdown studies, where Western blotting has been used to confirm the efficacy of siRNA-mediated silencing .

• Immunohistochemistry: URI1 antibodies have been validated for IHC applications in human lung cancer and human liver cancer tissues, with recommended dilutions ranging from 1:30 to 1:150 . This application is particularly important for analyzing URI1 expression in patient tumor samples and correlating with clinical outcomes.

• Research Models: URI1 antibodies have been successfully employed in studying HBV infection in both HepG2-NTCP cells and primary human hepatocytes (PHH) . They have also been used to investigate URI1's role in chemotherapy resistance mechanisms, particularly in studies examining URI1-induced ATM expression and resistance to cisplatin .

How can researchers distinguish between specific and non-specific URI1 antibody signals?

Distinguishing between specific and non-specific URI1 antibody signals requires several control strategies:

• Validation Controls: Include positive controls (cell lines with confirmed URI1 expression like 293T) and negative controls (URI1 knockdown samples or tissues known to have low URI1 expression) .

• Specificity Verification: In Western blotting experiments, verify specificity by confirming that siRNA-mediated URI1 knockdown results in corresponding reduction of the detected band. For example, studies have shown that URI1 mRNA levels were reduced by 40-70% in HepG2-NTCP cells five days post-siRNA transfection, with Western blot analysis confirming protein downregulation .

• Loading Controls: Always include appropriate loading controls such as GAPDH (for Western blotting) or reference genes (for qPCR). In published research, equal protein loading has been confirmed by detecting GAPDH or vinculin on the same membrane .

• Multiple Antibodies: When possible, use multiple antibodies targeting different epitopes of URI1 to confirm findings and rule out non-specific binding.

What are the established methods for URI1 knockdown in hepatocyte models?

URI1 knockdown in hepatocyte models has been successfully achieved through the following methodological approach:

• siRNA Transfection: Multiple siRNAs targeting URI1 have been validated, with transfection into both HepG2-NTCP cells and primary human hepatocytes (PHH) .

• Verification by RT-qPCR: URI1 knockdown efficiency should be verified using RT-qPCR. The Luna Universal One-Step RT-qPCR Kit (New England Biolabs) on a real-time PCR system has been used successfully. Primers targeting URI1 mRNA with normalization to GAPDH expression should be employed .

• Confirmation by Western Blotting: Protein level reduction should be confirmed via Western blotting. Cell lysates can be prepared using CelLytic M buffer, with 20 μg of lysate separated by SDS-PAGE on 4-15% precast protein gels. After transfer to nitrocellulose membrane, URI1 can be detected using specific primary antibody (e.g., Sigma-Aldrich #HPA071709) and an HRP-conjugated secondary antibody .

• Timing Considerations: In HepG2-NTCP cells, URI1 mRNA levels remained reduced by 40-70% compared to control five days post-infection, while all siRNAs tested reduced URI1 mRNA levels in PHH by approximately 70% seven days post-infection .

How can researchers investigate the relationship between URI1 and HBV infection?

To investigate the relationship between URI1 and HBV infection, researchers can employ these methodological approaches:

• Infection Models: Use HepG2-NTCP cells or primary human hepatocytes (PHH) infected with HBV. This system allows for studying the effects of URI1 manipulation on viral infection and replication .

• URI1 Manipulation: Implement URI1 silencing using siRNAs or overexpression using appropriate expression vectors prior to HBV infection .

• Viral Marker Assessment: After infection, assess viral markers such as HBc protein detection by Western blotting. Separate 10 μg of cell lysate by SDS-PAGE and detect HBc using specific antibodies (e.g., Gilead Sciences rabbit monoclonal antibody) .

• Infection Monitoring: Monitor HBV infection by quantifying viral genome equivalents (VGE) and detecting covalently closed circular DNA (cccDNA), which represents the stable nuclear form of the HBV genome .

• Clinical Correlation: Correlate URI1 expression levels with HBV markers in patient samples to establish clinical relevance .

What approaches can be used to study URI1's role in cancer treatment resistance?

To study URI1's role in treatment resistance, researchers can utilize the following approaches:

• Overexpression Studies: Create cell models with URI1 overexpression to investigate resistance mechanisms. Research has shown that URI1 overexpression induces ATM expression and resistance to cisplatin .

• Combination Therapy Models: Study the effects of URI1 manipulation in combination with cancer therapeutics. For example, URI1 overexpression has been shown to promote resistance to ferroptosis induced by tyrosine kinase inhibitors via stearoyl-CoA desaturase 1 (SCD1), while URI1 knockdown or SCD1 inhibition enhanced sensitivity to these inhibitors .

• Molecular Pathway Analysis: Investigate downstream effectors of URI1-mediated resistance. Transcriptome analysis has revealed that tumors with URI1 amplification display decreased transcription of genes encoding tumor suppressors and apoptotic regulators while showing increased expression of genes regulating oncogenesis, survival, and metastasis .

• Clinical Outcome Correlation: Analyze treatment response in patients with varying URI1 expression levels. Clinical studies have demonstrated that patients with URI1 amplification had poorer response to adjuvant treatment compared to control groups (P=0.013) .

How does URI1 contribute to transcriptional regulation through RNA polymerase II interactions?

URI1 functions as an RNA polymerase II subunit 5 mediating protein (RMP), playing a critical role in transcriptional regulation . Advanced research approaches to study this interaction include:

• Protein-Protein Interaction Studies: Implement co-immunoprecipitation assays to verify and characterize the interaction between URI1 and RPB5, as well as other components of the transcriptional machinery.

• Chromatin Immunoprecipitation (ChIP): Use ChIP assays with URI1 antibodies to identify genomic binding sites and understand its role in regulating specific gene promoters.

• Transcriptome Analysis: Perform RNA-seq on cells with normal versus altered URI1 expression to identify genes whose expression is regulated by URI1-mediated transcriptional control.

• Structure-Function Analysis: Create domain-specific mutations in URI1 to identify regions critical for RPB5 interaction and transcriptional regulation.

• Post-Translational Modification Analysis: Investigate how modifications of URI1 might regulate its transcriptional functions and interactions with RNA polymerase II components.

What is the significance of URI1's mitochondrial localization and potential therapeutic implications?

URI1 has been shown to localize to mitochondria, colocalizing with proteins including PFDN2, PFDN4, PPP1CC, RPS6KB1, and STAP1 . This mitochondrial localization may have significant implications for cancer metabolism and therapeutic targeting:

• Metabolic Regulation: Investigate URI1's role in mitochondrial function and cellular metabolism, particularly in the context of the altered metabolic requirements of cancer cells.

• Cell Death Pathways: Explore the relationship between mitochondrial URI1 and cell death mechanisms, particularly in response to chemotherapeutic agents.

• Therapeutic Targeting: Develop approaches to specifically target mitochondrial URI1 functions as a potential cancer therapy, particularly in HBV-associated HCC where URI1 has been proposed as a therapeutic target .

• Resistance Mechanisms: Study how mitochondrial URI1 contributes to treatment resistance, particularly through interactions with stearoyl-CoA desaturase 1 (SCD1) which has been implicated in tyrosine kinase inhibitor resistance .

• Biomarker Development: Investigate whether mitochondrial URI1 localization could serve as a biomarker for treatment response or disease progression.

What is the relationship between URI1 and DNA damage response pathways in cancer cells?

URI1 has been implicated in DNA damage response pathways, particularly through its relationship with ATM expression and cisplatin resistance . Advanced approaches to study this relationship include:

• DNA Damage Induction Models: Expose cells with varying URI1 expression to DNA-damaging agents to assess response patterns and repair efficiency.

• Pathway Analysis: Investigate the molecular mechanisms linking URI1 to DNA damage response, focusing on key regulators like ATM and p53.

• Cellular Imaging: Use immunofluorescence with URI1 antibodies to track subcellular localization changes following DNA damage, particularly examining potential recruitment to damage sites.

• Genetic Interaction Studies: Perform genetic screens to identify synthetic lethal interactions between URI1 and DNA repair pathway components.

• Therapeutic Exploitation: Explore how URI1 expression levels might be used to predict response to DNA-damaging agents in cancer therapy, potentially identifying patient subsets most likely to benefit from specific treatment approaches.