PRR11 Antibody

What is PRR11 and why is it significant in cancer research?

PRR11 is a newly identified oncogene associated with poor prognosis in several human cancers . Research has demonstrated its overexpression in multiple cancer types, including lung cancer, liver cancer, pancreatic cancer, and ovarian cancer . Its significance stems from its involvement in critical cellular processes, including:

• Cell cycle regulation, particularly in S phase progression

• F-actin assembly through interaction with the Arp2/3 complex

• Promotion of cell proliferation and migration

• Regulation of gene expression through interaction with transcription factors

PRR11's widespread oncogenic role across different cancer types makes it a valuable target for both diagnostic and therapeutic development, particularly as its expression levels correlate with poor patient outcomes in at least 10 types of cancers, including adrenocortical carcinoma, bladder cancer, kidney cancer, liver cancer, lung cancer, and pancreatic adenocarcinoma .

What are the recommended experimental techniques for detecting PRR11 using antibodies?

Several validated techniques have proven effective for PRR11 detection:

• Western Blotting: Recommended for quantitative analysis of PRR11 protein expression. Standard protocols using PRR11-specific antibodies (e.g., Thermo Fisher Scientific Cat. No. PA5113175, dilution 1:200) have shown reliable results . For optimal results, quantify proteins and resolve by 10% SDS-PAGE before transfer to PVDF membranes.

• Immunofluorescence: Effective for visualizing PRR11 localization and co-localization with other proteins. This technique has successfully demonstrated that PRR11 is primarily located in the nucleus , and can form meshwork structures when overexpressed .

• Co-Immunoprecipitation (Co-IP): Essential for investigating protein-protein interactions. Using magnetic IP/Co-IP kits (e.g., Pierce Classic Magnetic IP/Co-IP Kit), researchers have successfully demonstrated PRR11's interaction with Arp2/3 complex components and transcription factors like E2F1 .

• Chromatin Immunoprecipitation (ChIP-qPCR): Useful for studying PRR11's role in transcriptional regulation, particularly its interaction with promoter regions of target genes such as PTTG1 .

How should PRR11 knockdown experiments be designed and validated?

PRR11 knockdown experiments require careful design and validation:

• siRNA Design: For transient knockdown, use validated siRNA sequences targeting PRR11. The sequence 5′-ACGCAGGCCUUAAGGAGAATT-3′ has demonstrated efficacy in ovarian cancer cells .

• Lentiviral Approach: For stable knockdown, lentiviral expression plasmids containing siRNA targeting PRR11 provide consistent results. Commercial options are available from providers like GENECHEM Corporation .

• Validation Methods:

  • Western blotting: Primary validation method to confirm protein reduction

  • qPCR: Secondary validation to confirm mRNA reduction

  • Include appropriate controls (mock transfection or empty vector) to account for non-specific effects

• Functional Assays: After confirming knockdown efficiency, proceed with functional assays:

  • Proliferation assays (e.g., CCK-8 assay)

  • Migration assays (e.g., Transwell migration assay)

  • In vivo xenograft models for tumor growth assessment

Research has shown that successful PRR11 knockdown significantly reduces cancer cell proliferation both in short-term and long-term assays, and substantially decreases migration capabilities .

How does PRR11 interact with the Arp2/3 complex to drive F-actin assembly in cancer cells?

PRR11 drives F-actin assembly through direct interaction with the Arp2/3 complex, a key regulator of actin cytoskeleton dynamics. Experimental approaches to study this interaction include:

• Co-immunoprecipitation: This method has confirmed the physical interaction between PRR11 and Arp2/3 complex components. Both ectopically expressed Flag-tagged PRR11 and endogenous PRR11 have been shown to co-immunoprecipitate with Arp2 and Arp3 in H1299 lung cancer cells .

• Immunofluorescence Colocalization: PRR11 and Arp2 demonstrate significant colocalization in the cytoplasm, with PRR11 overexpression leading to characteristic meshwork structures. When investigating this phenomenon:

  • Use dual staining with both PRR11 and Arp2/3 antibodies

  • Analyze colocalization using confocal microscopy

  • Include Flag-PRR11 staining to distinguish between endogenous and exogenous PRR11

• Functional Studies: PRR11 overexpression has been shown to increase Arp2 and Arp3 protein levels, suggesting a role in regulating the abundance of Arp2/3 complex components in addition to their localization .

The experimental evidence indicates that PRR11 serves as a scaffold protein, recruiting the Arp2/3 complex to specific cellular locations, driving local F-actin assembly, and potentially enhancing cancer cell migration and invasion properties.

What molecular mechanisms underlie PRR11's regulation of PTTG1 expression and how does this impact cancer progression?

PRR11 regulates PTTG1 expression through a transcription factor-mediated mechanism that impacts cell cycle progression and cancer development. This complex interaction can be investigated through:

• Transcriptional Regulation Analysis:

  • ChIP-qPCR has demonstrated that PRR11 and E2F1 can both enrich at the E2F1 binding site (5′-TTTGGGGC-3′) of the PTTG1 promoter region (-256/-124) .

  • The E2F1 binding site prediction can be performed using the JASPAR online tool.

• Protein-Protein Interaction Studies:

  • Co-IP experiments have confirmed that PRR11 interacts with E2F1.

  • Western blot analysis has shown that PRR11 knockdown inhibits E2F1 expression, suggesting a feedback mechanism .

• Expression Correlation Analysis:

  • RNA-seq analysis after PRR11 knockdown shows PTTG1 downregulation.

  • This correlation is supported by TCGA data showing Spearman correlations >0.3 in 16/33 cancer types with ≥200 samples .

The mechanistic model suggests that PRR11 promotes PTTG1 expression by:

• Interacting with E2F1

• Binding to the E2F1 binding site in the PTTG1 promoter

• Enhancing transcriptional activation of PTTG1

• Consequently promoting cell cycle progression and sister chromatid separation

This represents a common mechanism by which PRR11 exerts its oncogenic effects across different cancer types, making it a potential pan-cancer therapeutic target.

How can contradictory findings about PRR11 signaling pathways in different cancer types be reconciled experimentally?

Several studies have reported different signaling pathways through which PRR11 exerts its effects in various cancer types, creating seemingly contradictory findings. These include:

• Akt/mTOR signaling in non-small-cell lung cancer

• p38 MAPK signaling in pancreatic cancer

• Wnt/β-catenin signaling in esophageal and hepatic carcinoma

To reconcile these findings experimentally:

• Multi-omics Approach:

  • Conduct parallel RNA-seq, phospho-proteomics, and protein-protein interaction studies in multiple cancer cell lines.

  • This approach addresses limitations of individual studies. For example, RNA-seq reveals gene expression changes but not phosphorylation status of signaling proteins .

• Context-Dependent Analysis:

  • Perform signaling pathway perturbation experiments across multiple cell lines simultaneously.

  • Use small molecule inhibitors of each pathway alongside PRR11 manipulation to identify direct versus indirect effects.

• Time-Course Experiments:

  • Analyze signaling events at multiple time points after PRR11 knockdown or overexpression.

  • This can distinguish between primary and secondary effects of PRR11 manipulation.

• Common Downstream Effectors:

  • Focus on cell cycle regulation, which appears to be a common downstream effect across different cancer types .

  • Use synchronized cell populations to precisely define PRR11's role in cell cycle progression.

Research has shown that despite differences in upstream signaling pathways, PRR11 consistently affects cell cycle progression across cancer types. This suggests that PRR11 may integrate signals from multiple pathways that converge on cell cycle regulation, particularly in S phase .

What are the methodological considerations for achieving optimal signal-to-noise ratio when using PRR11 antibodies in multimodal single-cell analyses?

When using PRR11 antibodies in advanced single-cell analyses, several methodological considerations can improve signal quality:

• Antibody Concentration Optimization:

  • For highly abundant epitopes, antibody concentration must be carefully titrated.

  • Low concentrations (0.0125 to 0.025 μg/mL) may be significantly affected by reduced staining volumes .

  • Consider a titration matrix experiment to determine optimal concentration.

• Cell Number Considerations:

  • Reducing cell numbers during staining (e.g., from 1×10^6 to 0.2×10^6 cells) can increase signal for antibodies used at low concentrations by reducing epitope competition .

  • This approach is particularly relevant for PRR11 detection in heterogeneous samples where PRR11-expressing cells may be rare.

• Validation Strategies:

  • Compare PRR11 antibody performance across multiple techniques (flow cytometry, immunofluorescence, Western blotting).

  • Validate antibody specificity using PRR11 knockdown controls.

  • Include isotype controls to account for non-specific binding.

• Signal Amplification Methods:

  • Consider secondary antibody approaches or tyramide signal amplification for low-abundance targets.

  • For multi-modal analyses, ensure that PRR11 detection does not interfere with other measurement modalities.

• Sample Preparation Optimization:

  • Test different fixation and permeabilization protocols to maximize epitope accessibility.

  • For nuclear-localized PRR11, ensure proper nuclear permeabilization.

When working with PRR11 antibodies in single-cell experiments, these methodological considerations are essential, as they directly influence detection sensitivity and specificity, particularly when studying heterogeneous tissues where PRR11 expression may vary significantly between cell populations.

How can protein-protein interaction networks of PRR11 be comprehensively mapped to identify novel therapeutic targets?

Mapping PRR11's interaction network requires systematic approaches:

• Proximity-Based Labeling Techniques:

  • BioID or TurboID approaches: Fuse PRR11 with a biotin ligase to label proximal proteins

  • APEX2 proximity labeling: Provides temporal resolution of interactions

  • These methods capture transient and weak interactions that conventional Co-IP might miss

• Mass Spectrometry-Based Approaches:

  • Quantitative interaction proteomics using SILAC or TMT labeling

  • Compare PRR11 interactomes across multiple cancer types to identify common versus tissue-specific interactions

  • Focus on specific cellular compartments, particularly nuclear interactions given PRR11's nuclear localization

• Domain-Specific Interaction Mapping:

  • Create a series of PRR11 deletion mutants to map which domains are responsible for specific interactions

  • This approach has proven valuable for understanding which regions of PRR11 mediate its interaction with E2F1 and the Arp2/3 complex

• Validation Strategies:

  • Confirm key interactions through multiple methods (Co-IP, proximity ligation assay, FRET)

  • Use CRISPR-Cas9 to disrupt interaction sites and assess functional consequences

  • Develop peptide inhibitors of key interactions for therapeutic validation

• Computational Analysis:

  • Integrate interaction data with existing cancer dependency databases

  • Prioritize interactions that correlate with cancer vulnerability

  • Build predictive models of PRR11-dependent pathways

This comprehensive mapping approach has already identified important PRR11 interaction partners (Arp2/3 complex, E2F1) and could reveal additional targetable nodes in PRR11-dependent cancer pathways.