PRUNE2 Antibody

What is PRUNE2 and what is its role in cancer biology?

PRUNE2 (prune homolog 2) is a protein with multiple functional domains including BCH, DHHA2, and PPX1. The protein plays a crucial role in regulating cellular processes including morphogenesis, differentiation, motility and apoptosis by interacting with components of signaling networks such as Rho, Ras and MAPK pathways . The calculated molecular weight of this protein is approximately 340 kDa, although commercial antibodies typically recognize a 140-150 kDa band, which likely represents an isoform or cleaved fragment of PRUNE2 .

PRUNE2 functions as a tumor suppressor in multiple cancer types:

• In prostate cancer, it is regulated by PCA3 (prostate cancer antigen 3), a long non-coding RNA that forms a double-stranded RNA with PRUNE2 pre-mRNA, leading to ADAR-dependent adenosine-to-inosine RNA editing

• In colorectal cancer, PRUNE2 overexpression decreases cell proliferation and invasion, increases apoptosis, arrests cell cycle, and reduces tumorigenicity

• In neuroblastoma, higher PRUNE2 expression correlates with favorable prognosis

When investigating PRUNE2 in cancer contexts, researchers should consider both transcriptional regulation and post-translational modifications that may affect protein function.

What experimental applications can PRUNE2 antibodies be used for?

PRUNE2 antibodies have been validated for multiple research applications:

For immunohistochemistry applications, antigen retrieval may be performed with either:

• TE buffer pH 9.0 (recommended)

• Citrate buffer pH 6.0 (alternative)

When performing IHC, successful detection has been reported in human liver cancer tissue . For optimal results, researchers should titrate the antibody concentration in each experimental system and include appropriate positive and negative controls to validate specificity.

Why does the PRUNE2 antibody detect a band at 140-150 kDa when the calculated molecular weight is 340 kDa?

This apparent molecular weight discrepancy is a common source of confusion in PRUNE2 research. While the calculated molecular weight based on the full protein sequence is approximately 340-341 kDa, commercial antibodies typically recognize a 140-150 kDa band . This difference is due to one of several possibilities:

• Isoform detection: PRUNE2 may exist in multiple isoforms due to alternative splicing, and the antibody may be detecting a specific isoform

• Protein processing: PRUNE2 may undergo post-translational processing, resulting in cleaved fragments

• Protein degradation: The full-length protein may be unstable during sample preparation

When validating PRUNE2 antibodies in your experimental system, consider running:

• Positive controls from tissues known to express PRUNE2 (e.g., brain tissue)

• Negative controls using PRUNE2 knockdown or knockout samples

• Recombinant PRUNE2 protein standards if available

This validation is particularly important when studying different tissue types, as expression patterns may vary significantly between neural tissues (where expression is typically high) and other tissues where expression may be lower .

What is the relationship between PRUNE2 and PCA3 in prostate cancer?

The PRUNE2/PCA3 relationship represents a fascinating regulatory mechanism in prostate cancer biology:

• Genomic relationship: PCA3 (prostate cancer antigen 3) is a long non-coding RNA located within intron 6 of the PRUNE2 gene and transcribed in the antisense direction

• Regulatory mechanism: PCA3 forms a double-stranded RNA with PRUNE2 pre-mRNA, which undergoes adenosine deaminase acting on RNA (ADAR)-dependent adenosine-to-inosine RNA editing

• Expression pattern: In prostate cancer tissues, PCA3 is typically upregulated while PRUNE2 is downregulated compared to adjacent normal prostate tissue

• Functional consequences:

  • PRUNE2 overexpression decreases cell proliferation

  • PRUNE2 silencing increases cell proliferation

  • PCA3 and PRUNE2 elicit opposite effects on tumor growth in mouse models

• Clinical relevance: While the reciprocal expression pattern is consistent across tumor grades and stages, studies have not found an association between the relative expression levels of PCA3 or PRUNE2 and time to disease recurrence

This regulatory axis highlights the importance of considering both coding and non-coding elements when studying PRUNE2 expression in prostate cancer contexts.

What are the recommended methods for validating PRUNE2 antibody specificity?

Rigorous validation of PRUNE2 antibodies is essential due to the complexity of this protein (multiple domains, potential isoforms, and large size). Comprehensive validation should include:

• Genetic validation approaches:

  • PRUNE2 knockdown using siRNA or shRNA

  • PRUNE2 knockout using CRISPR-Cas9

  • PRUNE2 overexpression systems

  For example, in colorectal cancer research, SW620 and HT29 cells have been successfully transfected with PRUNE2 shRNA or PRUNE2/pcDNA3.1 vector to modulate expression levels .

• Biochemical validation:

  • Peptide competition assays

  • Detection of recombinant protein

  • Mass spectrometry verification of immunoprecipitated proteins

• Cross-validation with multiple antibodies:

  • Compare results from antibodies targeting different epitopes

  • Evaluate specificity across different applications (WB, IHC, IF)

• Cell/tissue expression profiling:

  • Compare protein detection with known mRNA expression patterns

  • Test in tissues with known high expression (brain, cerebellum, spinal cord)

  • Test in cell lines with varying expression (neuroblastoma, rhabdomyosarcoma, melanoma, and osteosarcoma cell lines show high expression; liver, breast, thyroid, and colon cancer cell lines show low expression)

When publishing PRUNE2 research, include comprehensive antibody validation data to enhance reproducibility and reliability of findings.

How can researchers accurately quantify PRUNE2 expression in clinical samples?

Accurate quantification of PRUNE2 expression in clinical samples poses several technical challenges that require specialized approaches:

• RNA-based quantification:

  • qRT-PCR with multiple primer sets targeting different exons

  • Use of appropriate housekeeping genes (multiple controls recommended)

  • RNA-Seq analysis with attention to isoform-specific expression

  In published studies, researchers have used TaqMan gene expression assays with multiple control genes for validation. For example, in one study, nine duplex mixes for PRUNE2 (PR1C1, PR1C2, PR1C3, etc.) were used with three different control genes to ensure robust quantification .

• Protein-based quantification:

  • Western blot analysis with normalization to loading controls

  • Densitometry analysis of immunohistochemistry

  • Multi-parameter flow cytometry for cell-based studies

• Tissue preparation considerations:

  • Microdissection of FFPE sections to isolate specific cell populations

  • RNA quality assessment (e.g., using Bioanalyzer) to ensure reliable quantification

  • DNase treatment to remove genomic DNA contamination

• Data analysis approaches:

  • Use of multiple technical and biological replicates

  • Statistical methods appropriate for clinical samples (e.g., Wilcoxon signed rank test for paired tumor and normal tissues)

  • Consider analyzing the PRUNE2/PCA3 expression ratio rather than individual gene levels

For clinical studies, researchers should report detailed methodologies and consider the impact of pre-analytical variables (tissue collection, fixation time, storage conditions) on PRUNE2 quantification.

What molecular mechanisms underlie PRUNE2's tumor suppressive function?

The tumor suppressive properties of PRUNE2 operate through multiple molecular mechanisms:

• Modulation of signaling pathways:

  • The BCH domain of PRUNE2 can inhibit the Rho family of proteins, small GTPases involved in cell transformation, migration, metastasis, and cell cycle progression

  • PRUNE2 may interact with components of Ras and MAPK signaling networks

• Regulation of apoptosis:

  • PRUNE2 overexpression increases cell apoptosis in colorectal cancer cells

  • It increases expression of pro-apoptotic genes and decreases expression of anti-apoptotic proteins

• Cell cycle regulation:

  • PRUNE2 overexpression arrests the cell cycle in colorectal cancer models

• Genomic alterations:

  • Loss-of-function mutations in PRUNE2 have been described in several tumor types, including:

    • Germline and somatic mutations in parathyroid cancer

    • Somatic mutations in solid papillary carcinoma

    • Inactivating mutations in Merkel cell carcinoma

• Post-transcriptional regulation:

  • The regulatory interaction with PCA3 lncRNA represents a unique mechanism of tumor suppressor control through RNA editing

When designing experiments to study PRUNE2's molecular functions, researchers should consider both genetic approaches (manipulation of expression) and biochemical techniques (protein interaction studies, signaling pathway analysis) to comprehensively characterize its role in specific cancer contexts.

How does the PRUNE2/PCA3 regulatory axis function at the molecular level?

The PRUNE2/PCA3 regulatory axis represents a sophisticated example of gene regulation through RNA-RNA interactions:

• Genomic architecture:

  • PCA3 is embedded within intron 6 of PRUNE2

  • PCA3 is transcribed in the antisense direction relative to PRUNE2

• Double-stranded RNA formation:

  • PCA3 lncRNA forms a double-stranded RNA structure with the complementary sequence in PRUNE2 pre-mRNA

  • This dsRNA formation is critical for the regulatory function

• RNA editing mechanism:

  • The double-stranded RNA undergoes adenosine deaminase acting on RNA (ADAR)-dependent adenosine-to-inosine RNA editing

  • This editing process affects PRUNE2 expression and processing

• Experimental evidence for this mechanism:

  • Ectopic PCA3 expression decreases endogenous PRUNE2 protein, pre-mRNA, and mRNA levels

  • PCA3 silencing increases PRUNE2 levels

  • Expression of a PRUNE2 construct lacking complementarity to PCA3 is not affected by PCA3 levels

  • Expression of intron6-PRUNE2 (containing PCA3-complementary sequence but no protein-coding sequence) can sequester PCA3 and increase endogenous PRUNE2 mRNA in the cytoplasm

• Cell type specificity:

  • This regulatory mechanism is particularly active in androgen-dependent prostate cancer cells (e.g., LNCaP) compared to androgen-independent cells (DU145 and PC3)

To study this mechanism, researchers can employ approaches such as RNA immunoprecipitation, RNA editing site analysis, subcellular fractionation, and targeted manipulation of the complementary RNA regions.

What are the technical challenges in studying PRUNE2 expression in different experimental systems?

Investigating PRUNE2 presents several technical challenges that researchers should address with specialized approaches:

• Protein size and detection issues:

  • The full-length PRUNE2 protein is exceptionally large (340-341 kDa)

  • Commercial antibodies typically detect a 140-150 kDa band, complicating data interpretation

  • Protein transfer efficiency may be limited for very large proteins in Western blot applications

• Multiple isoforms and processing variants:

  • PRUNE2 exists in multiple isoforms

  • Post-translational processing may generate different protein fragments

  • Researchers should consider isoform-specific detection methods

• Spatial expression patterns:

  • Expression varies significantly across tissues (high in neural tissues)

  • Cell-type-specific expression within heterogeneous tissue samples requires microdissection or single-cell approaches

• Regulatory complexities:

  • The antisense PCA3 lncRNA regulation adds complexity to expression analysis

  • RNA editing events need specialized detection methods

  • Consider analyzing both PRUNE2 and PCA3 simultaneously for complete understanding

• Experimental models:

  • Different cell lines show variable PRUNE2 expression levels

  • LNCaP cells (androgen-dependent) show higher expression than DU145 and PC3 (androgen-independent) cells

  • Expression may be altered by culture conditions and passage number

To address these challenges, researchers should implement:

• Multiple detection methods (RNA and protein-based)

• Isoform-specific analysis approaches

• Careful selection of experimental models based on expression patterns

• Comprehensive controls for antibody specificity and knockdown efficiency

What are the optimal sample preparation protocols for PRUNE2 immunohistochemistry?

For successful PRUNE2 immunohistochemistry, the following optimized protocol has been validated in research settings:

• Tissue fixation and processing:

  • Fix tissues in 10% neutral buffered formalin

  • Process and embed in paraffin following standard protocols

  • Section tissues at 4-5 μm thickness

• Antigen retrieval (critical for PRUNE2 detection):

  • Primary recommendation: TE buffer pH 9.0

  • Alternative approach: Citrate buffer pH 6.0 at 100°C for 15 min at 80 kPa

  • Complete antigen retrieval is essential for accurate detection

• Blocking and antibody incubation:

  • Block endogenous peroxidase/phosphatase activity with 3% H₂O₂ for 10 min at room temperature

  • Block with 5% goat serum in PBS for 15 min at room temperature

  • Incubate with PRUNE2 antibody (1:50-1:500 dilution, optimize for your specific tissue) overnight at 4°C

  • Incubate with HRP-conjugated secondary antibody (e.g., 1:200 dilution) for 1.5 h at room temperature

• Visualization and counterstaining:

  • Visualize using DAB detection system

  • Counterstain with 0.5% hematoxylin for 5-10 min at room temperature

  • Brown staining indicates positive PRUNE2 expression; blue staining indicates nuclei

• Quantification approaches:

  • Quantify positive expression using image analysis software (e.g., ImageJ)

  • Consider both staining intensity and percentage of positive cells

Including both positive controls (tissues known to express PRUNE2) and negative controls (antibody omission and PRUNE2-low tissues) is essential for validating staining specificity.

How should researchers design experiments to study the functional effects of PRUNE2?

Designing robust experiments to study PRUNE2 function requires careful consideration of several methodological aspects:

• Genetic manipulation approaches:

  • Overexpression systems: Use PRUNE2/pcDNA3.1 vectors for transient expression

  • Knockdown strategies: Apply PRUNE2-specific shRNA or siRNA

  • Stable cell lines: Generate cells with inducible PRUNE2 expression for long-term studies

  • Partial constructs: Consider expressing specific domains to dissect functional regions

• Functional assays to assess tumor suppressive activity:

  • Proliferation: Cell Counting Kit-8 (CCK-8) assay

  • Colony formation: Assess clonogenic potential

  • Apoptosis: Flow cytometry with annexin V/PI staining

  • Cell cycle: Flow cytometry with PI staining

  • Invasion: Transwell assay

  • In vivo tumorigenicity: Mouse xenograft models

• Molecular mechanism studies:

  • Protein interaction partners: Co-immunoprecipitation, proximity ligation assays

  • Signaling pathway analysis: Western blot for key signaling molecules

  • RNA regulation: RNA immunoprecipitation, RNA editing site analysis

• Experimental design considerations:

  • Include multiple cell lines with varying endogenous PRUNE2 expression

  • Implement appropriate controls (empty vector, non-targeting shRNA)

  • Use rescue experiments to confirm specificity of observed effects

  • Consider the impact of PCA3 expression when working with prostate cancer models

• Data analysis approaches:

  • Normalize data to appropriate controls

  • Use statistical methods suitable for each assay

  • Consider the temporal aspects of PRUNE2 effects

A comprehensive experimental approach examining both phenotypic effects and underlying molecular mechanisms will provide the most valuable insights into PRUNE2 function in cancer biology.

What approaches can address conflicting findings regarding PRUNE2 expression in different cancer types?

The literature contains some apparently contradictory findings regarding PRUNE2 expression patterns and functions. Researchers can employ several strategies to address these inconsistencies:

• Comprehensive expression profiling:

  • Analyze multiple datasets across cancer types (TCGA, GEO, etc.)

  • Stratify by cancer subtypes, stages, and molecular features

  • Consider both mRNA and protein expression patterns

  • Analyze multiple patient cohorts (discovery and validation cohorts)

• Methodological standardization:

  • Employ multiple detection methods (qRT-PCR, RNA-Seq, IHC, Western blot)

  • Use clearly defined antibodies with validation data

  • Implement consistent sample processing protocols

  • Report detailed methodologies to enable reproduction

• Context-specific analysis:

  • Consider cell-type specificity within heterogeneous samples

  • Analyze expression in relation to tumor microenvironment

  • Evaluate the impact of genetic background and concurrent mutations

  • Assess the influence of hormonal factors (particularly in prostate cancer)

• Resolution approaches for specific contradictions:

  • For conflicting findings regarding androgen regulation in prostate cancer, one study reported PRUNE2 as androgen-inducible in a small sample set, while a larger study found no androgen induction

  • Comparative analysis between androgen-dependent (LNCaP) and androgen-independent (DU145, PC3) cell lines can help resolve such contradictions

• Integrated analysis:

  • Consider PRUNE2 in the context of its regulatory partners (e.g., PCA3)

  • Evaluate isoform-specific expression patterns

  • Assess post-translational modifications and protein stability

By implementing these approaches, researchers can develop a more nuanced understanding of PRUNE2's role across different cancer contexts and reconcile apparently contradictory findings in the literature.

PRUNE2 Antibody: Frequently Asked Questions for Researchers

What is PRUNE2 and what is its role in cancer biology?

PRUNE2 (prune homolog 2) is a protein with multiple functional domains including BCH, DHHA2, and PPX1. The protein plays a crucial role in regulating cellular processes including morphogenesis, differentiation, motility and apoptosis by interacting with components of signaling networks such as Rho, Ras and MAPK pathways . The calculated molecular weight of this protein is approximately 340 kDa, although commercial antibodies typically recognize a 140-150 kDa band, which likely represents an isoform or cleaved fragment of PRUNE2 .

PRUNE2 functions as a tumor suppressor in multiple cancer types:

• In prostate cancer, it is regulated by PCA3 (prostate cancer antigen 3), a long non-coding RNA that forms a double-stranded RNA with PRUNE2 pre-mRNA, leading to ADAR-dependent adenosine-to-inosine RNA editing

• In colorectal cancer, PRUNE2 overexpression decreases cell proliferation and invasion, increases apoptosis, arrests cell cycle, and reduces tumorigenicity

• In neuroblastoma, higher PRUNE2 expression correlates with favorable prognosis

When investigating PRUNE2 in cancer contexts, researchers should consider both transcriptional regulation and post-translational modifications that may affect protein function.

What experimental applications can PRUNE2 antibodies be used for?

PRUNE2 antibodies have been validated for multiple research applications:

For immunohistochemistry applications, antigen retrieval may be performed with either:

• TE buffer pH 9.0 (recommended)

• Citrate buffer pH 6.0 (alternative)

When performing IHC, successful detection has been reported in human liver cancer tissue . For optimal results, researchers should titrate the antibody concentration in each experimental system and include appropriate positive and negative controls to validate specificity.

Why does the PRUNE2 antibody detect a band at 140-150 kDa when the calculated molecular weight is 340 kDa?

This apparent molecular weight discrepancy is a common source of confusion in PRUNE2 research. While the calculated molecular weight based on the full protein sequence is approximately 340-341 kDa, commercial antibodies typically recognize a 140-150 kDa band . This difference is due to one of several possibilities:

• Isoform detection: PRUNE2 may exist in multiple isoforms due to alternative splicing, and the antibody may be detecting a specific isoform

• Protein processing: PRUNE2 may undergo post-translational processing, resulting in cleaved fragments

• Protein degradation: The full-length protein may be unstable during sample preparation

When validating PRUNE2 antibodies in your experimental system, consider running:

• Positive controls from tissues known to express PRUNE2 (e.g., brain tissue)

• Negative controls using PRUNE2 knockdown or knockout samples

• Recombinant PRUNE2 protein standards if available

This validation is particularly important when studying different tissue types, as expression patterns may vary significantly between neural tissues (where expression is typically high) and other tissues where expression may be lower .

What is the relationship between PRUNE2 and PCA3 in prostate cancer?

The PRUNE2/PCA3 relationship represents a fascinating regulatory mechanism in prostate cancer biology:

• Genomic relationship: PCA3 (prostate cancer antigen 3) is a long non-coding RNA located within intron 6 of the PRUNE2 gene and transcribed in the antisense direction

• Regulatory mechanism: PCA3 forms a double-stranded RNA with PRUNE2 pre-mRNA, which undergoes adenosine deaminase acting on RNA (ADAR)-dependent adenosine-to-inosine RNA editing

• Expression pattern: In prostate cancer tissues, PCA3 is typically upregulated while PRUNE2 is downregulated compared to adjacent normal prostate tissue

• Functional consequences:

  • PRUNE2 overexpression decreases cell proliferation

  • PRUNE2 silencing increases cell proliferation

  • PCA3 and PRUNE2 elicit opposite effects on tumor growth in mouse models

• Clinical relevance: While the reciprocal expression pattern is consistent across tumor grades and stages, studies have not found an association between the relative expression levels of PCA3 or PRUNE2 and time to disease recurrence

This regulatory axis highlights the importance of considering both coding and non-coding elements when studying PRUNE2 expression in prostate cancer contexts.

What are the recommended methods for validating PRUNE2 antibody specificity?

Rigorous validation of PRUNE2 antibodies is essential due to the complexity of this protein (multiple domains, potential isoforms, and large size). Comprehensive validation should include:

• Genetic validation approaches:

  • PRUNE2 knockdown using siRNA or shRNA

  • PRUNE2 knockout using CRISPR-Cas9

  • PRUNE2 overexpression systems

  For example, in colorectal cancer research, SW620 and HT29 cells have been successfully transfected with PRUNE2 shRNA or PRUNE2/pcDNA3.1 vector to modulate expression levels .

• Biochemical validation:

  • Peptide competition assays

  • Detection of recombinant protein

  • Mass spectrometry verification of immunoprecipitated proteins

• Cross-validation with multiple antibodies:

  • Compare results from antibodies targeting different epitopes

  • Evaluate specificity across different applications (WB, IHC, IF)

• Cell/tissue expression profiling:

  • Compare protein detection with known mRNA expression patterns

  • Test in tissues with known high expression (brain, cerebellum, spinal cord)

  • Test in cell lines with varying expression (neuroblastoma, rhabdomyosarcoma, melanoma, and osteosarcoma cell lines show high expression; liver, breast, thyroid, and colon cancer cell lines show low expression)

When publishing PRUNE2 research, include comprehensive antibody validation data to enhance reproducibility and reliability of findings.

How can researchers accurately quantify PRUNE2 expression in clinical samples?

Accurate quantification of PRUNE2 expression in clinical samples poses several technical challenges that require specialized approaches:

• RNA-based quantification:

  • qRT-PCR with multiple primer sets targeting different exons

  • Use of appropriate housekeeping genes (multiple controls recommended)

  • RNA-Seq analysis with attention to isoform-specific expression

  In published studies, researchers have used TaqMan gene expression assays with multiple control genes for validation. For example, in one study, nine duplex mixes for PRUNE2 (PR1C1, PR1C2, PR1C3, etc.) were used with three different control genes to ensure robust quantification .

• Protein-based quantification:

  • Western blot analysis with normalization to loading controls

  • Densitometry analysis of immunohistochemistry

  • Multi-parameter flow cytometry for cell-based studies

• Tissue preparation considerations:

  • Microdissection of FFPE sections to isolate specific cell populations

  • RNA quality assessment (e.g., using Bioanalyzer) to ensure reliable quantification

  • DNase treatment to remove genomic DNA contamination

• Data analysis approaches:

  • Use of multiple technical and biological replicates

  • Statistical methods appropriate for clinical samples (e.g., Wilcoxon signed rank test for paired tumor and normal tissues)

  • Consider analyzing the PRUNE2/PCA3 expression ratio rather than individual gene levels

For clinical studies, researchers should report detailed methodologies and consider the impact of pre-analytical variables (tissue collection, fixation time, storage conditions) on PRUNE2 quantification.

What molecular mechanisms underlie PRUNE2's tumor suppressive function?

The tumor suppressive properties of PRUNE2 operate through multiple molecular mechanisms:

• Modulation of signaling pathways:

  • The BCH domain of PRUNE2 can inhibit the Rho family of proteins, small GTPases involved in cell transformation, migration, metastasis, and cell cycle progression

  • PRUNE2 may interact with components of Ras and MAPK signaling networks

• Regulation of apoptosis:

  • PRUNE2 overexpression increases cell apoptosis in colorectal cancer cells

  • It increases expression of pro-apoptotic genes and decreases expression of anti-apoptotic proteins

• Cell cycle regulation:

  • PRUNE2 overexpression arrests the cell cycle in colorectal cancer models

• Genomic alterations:

  • Loss-of-function mutations in PRUNE2 have been described in several tumor types, including:

    • Germline and somatic mutations in parathyroid cancer

    • Somatic mutations in solid papillary carcinoma

    • Inactivating mutations in Merkel cell carcinoma

• Post-transcriptional regulation:

  • The regulatory interaction with PCA3 lncRNA represents a unique mechanism of tumor suppressor control through RNA editing

When designing experiments to study PRUNE2's molecular functions, researchers should consider both genetic approaches (manipulation of expression) and biochemical techniques (protein interaction studies, signaling pathway analysis) to comprehensively characterize its role in specific cancer contexts.

What are the optimal sample preparation protocols for PRUNE2 immunohistochemistry?

For successful PRUNE2 immunohistochemistry, the following optimized protocol has been validated in research settings:

• Tissue fixation and processing:

  • Fix tissues in 10% neutral buffered formalin

  • Process and embed in paraffin following standard protocols

  • Section tissues at 4-5 μm thickness

• Antigen retrieval (critical for PRUNE2 detection):

  • Primary recommendation: TE buffer pH 9.0

  • Alternative approach: Citrate buffer pH 6.0 at 100°C for 15 min at 80 kPa

  • Complete antigen retrieval is essential for accurate detection

• Blocking and antibody incubation:

  • Block endogenous peroxidase/phosphatase activity with 3% H₂O₂ for 10 min at room temperature

  • Block with 5% goat serum in PBS for 15 min at room temperature

  • Incubate with PRUNE2 antibody (1:50-1:500 dilution, optimize for your specific tissue) overnight at 4°C

  • Incubate with HRP-conjugated secondary antibody (e.g., 1:200 dilution) for 1.5 h at room temperature

• Visualization and counterstaining:

  • Visualize using DAB detection system

  • Counterstain with 0.5% hematoxylin for 5-10 min at room temperature

  • Brown staining indicates positive PRUNE2 expression; blue staining indicates nuclei

• Quantification approaches:

  • Quantify positive expression using image analysis software (e.g., ImageJ)

  • Consider both staining intensity and percentage of positive cells

Including both positive controls (tissues known to express PRUNE2) and negative controls (antibody omission and PRUNE2-low tissues) is essential for validating staining specificity.

How should researchers design experiments to study the functional effects of PRUNE2?

Designing robust experiments to study PRUNE2 function requires careful consideration of several methodological aspects:

• Genetic manipulation approaches:

  • Overexpression systems: Use PRUNE2/pcDNA3.1 vectors for transient expression

  • Knockdown strategies: Apply PRUNE2-specific shRNA or siRNA

  • Stable cell lines: Generate cells with inducible PRUNE2 expression for long-term studies

  • Partial constructs: Consider expressing specific domains to dissect functional regions

• Functional assays to assess tumor suppressive activity:

  • Proliferation: Cell Counting Kit-8 (CCK-8) assay

  • Colony formation: Assess clonogenic potential

  • Apoptosis: Flow cytometry with annexin V/PI staining

  • Cell cycle: Flow cytometry with PI staining

  • Invasion: Transwell assay

  • In vivo tumorigenicity: Mouse xenograft models

• Molecular mechanism studies:

  • Protein interaction partners: Co-immunoprecipitation, proximity ligation assays

  • Signaling pathway analysis: Western blot for key signaling molecules

  • RNA regulation: RNA immunoprecipitation, RNA editing site analysis

• Experimental design considerations:

  • Include multiple cell lines with varying endogenous PRUNE2 expression

  • Implement appropriate controls (empty vector, non-targeting shRNA)

  • Use rescue experiments to confirm specificity of observed effects

  • Consider the impact of PCA3 expression when working with prostate cancer models

• Data analysis approaches:

  • Normalize data to appropriate controls

  • Use statistical methods suitable for each assay

  • Consider the temporal aspects of PRUNE2 effects

A comprehensive experimental approach examining both phenotypic effects and underlying molecular mechanisms will provide the most valuable insights into PRUNE2 function in cancer biology.