PPIH Antibody

What is PPIH and what are its primary cellular functions?

PPIH (Peptidyl-prolyl cis-trans isomerase H) is an enzyme that catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides, thereby assisting in protein folding processes . Beyond this enzymatic activity, PPIH plays crucial roles in:

• Pre-mRNA splicing mechanisms

• Assembly of the U4/U5/U6 tri-snRNP complex, a fundamental building block of the spliceosome

• Potential chaperone functions in cellular pathways

The protein is also known by several alternative names, including CYP20, CYPH, Small nuclear ribonucleoprotein particle-specific cyclophilin H, and U-snRNP-associated cyclophilin SnuCyp-20 .

What are the most common applications for PPIH antibodies in research?

PPIH antibodies have demonstrated utility in multiple experimental applications, particularly:

• Western blotting (WB): For detecting PPIH protein expression in cell lysates and tissue homogenates

• Immunohistochemistry on paraffin-embedded tissues (IHC-P): For visualizing PPIH expression patterns in normal and pathological tissue sections

• Immunocytochemistry with immunofluorescence (ICC-IF): For subcellular localization studies

These methodologies have been instrumental in recent breakthrough studies examining PPIH's role in various cancers, particularly cholangiocarcinoma and hepatocellular carcinoma .

What is the predicted molecular weight of PPIH and how does this impact antibody validation?

The predicted band size for PPIH is approximately 19 kDa . When validating PPIH antibodies, researchers should consider:

• Confirming band specificity at this molecular weight in Western blot applications

• Using appropriate positive controls (e.g., PC-3 human prostate adenocarcinoma cell line lysates have been validated for this purpose)

• Implementing secondary antibody controls to eliminate non-specific binding (goat polyclonal to rabbit IgG at 1/10000 dilution has been demonstrated as effective)

What tissue types are most suitable for PPIH expression studies?

Based on immunohistochemical analyses, several tissues have proven valuable for PPIH expression studies:

• Human tonsil tissue has been successfully used for paraffin-embedded IHC protocols

• Liver tissues (both normal and cancerous) show differential PPIH expression patterns relevant to hepatocellular carcinoma research

• Bile duct tissues for cholangiocarcinoma investigations

When designing experiments, researchers should select appropriate tissue models based on their specific research questions regarding PPIH function or expression.

What are the optimal conditions for using PPIH antibodies in Western blot applications?

For Western blot applications with PPIH antibodies, the following methodological parameters have demonstrated optimal results:

• Dilution: A 1/500 dilution of anti-PPIH antibody (e.g., ab235595) has shown effective detection sensitivity

• Sample preparation: Whole cell lysates are generally preferable to subcellular fractions

• Sample type: Human prostate adenocarcinoma cell line (PC-3) lysates have been validated as positive controls

• Secondary antibody: Goat polyclonal to rabbit IgG at 1/10000 dilution provides optimal signal-to-noise ratio

• Expected band size: 19 kDa (verification of this band size is crucial for result validation)

Researchers should optimize these parameters based on their specific experimental conditions and antibody batches.

How should researchers design IHC-P protocols for PPIH detection in tumor tissues?

For effective PPIH detection in paraffin-embedded tumor tissues, the following protocol has been validated:

• Deparaffinization and rehydration of tissue sections

• Antigen retrieval (heat-induced epitope retrieval is recommended)

• Blocking of endogenous peroxidase activity

• Primary antibody incubation: Anti-PPIH antibody at 1:100 dilution, overnight at 4°C

• Secondary antibody application: Biotin-labeled goat anti-rabbit IgG, followed by streptavidin-biotin-peroxidase complex at room temperature for 30 minutes

• Detection: 3,3'-diaminobenzidine tetrachloride (DAB) with hematoxylin counterstaining

• Dehydration with ethanol, clearing with xylene, and mounting

Positive PPIH expression is typically indicated by cytoplasmic staining of yellow or brownish-yellow color .

What controls should be included when evaluating PPIH expression in clinical samples?

When analyzing PPIH expression in clinical samples, the following controls are essential:

• Positive tissue controls: Validated tissues known to express PPIH (e.g., tonsil)

• Negative controls: Primary antibody omission or isotype controls to assess non-specific binding

• Internal controls: Adjacent normal tissues whenever possible for comparative analysis

• Technical replicates: Multiple tissue sections should be assessed to account for intratumoral heterogeneity

Additionally, researchers should consider implementing standardized scoring systems for PPIH expression that account for both staining intensity and percentage of positive cells to facilitate cross-study comparisons.

What is the diagnostic value of PPIH as a biomarker in cholangiocarcinoma?

Recent research has established significant diagnostic value for PPIH in cholangiocarcinoma (CHOL):

• Receiver Operating Characteristic (ROC) curve analysis revealed impressive area under the curve (AUC) values:

  • GSE32958 dataset: AUC = 0.914 (95% CI: 0.800–0.995)

  • GSE76311 dataset: AUC = 0.963 (95% CI: 0.936–0.984)

These values indicate exceptional discriminatory ability between CHOL and normal tissues, surpassing the diagnostic performance of other potential biomarkers including PTPRS, UBE2C, CEACAM5, and THBS2 .

Immunohistochemical validation has confirmed significantly elevated PPIH protein levels in CHOL tissues compared to adjacent normal tissues, further supporting its potential as a diagnostic biomarker .

How does PPIH expression correlate with patient prognosis in liver cancers?

The prognostic significance of PPIH expression has been investigated in multiple liver cancers:

These findings suggest that PPIH expression levels may serve as a valuable prognostic indicator, though additional validation in larger cohorts is needed.

What molecular pathways are implicated in PPIH-mediated tumor progression?

Research has identified several key molecular pathways through which PPIH may influence tumor progression:

• Cell cycle regulation: PPIH overexpression appears to impact cell cycle control mechanisms, potentially promoting tumor cell proliferation

• Spliceosome function: Given PPIH's role in spliceosome assembly, aberrant splicing patterns may contribute to oncogenic processes

• Immune modulation: PPIH expression is associated with altered immune cell infiltration in tumor microenvironments

Gene enrichment analyses have specifically implicated PPIH in these pathways, suggesting multiple potential mechanisms through which PPIH may influence tumor development and progression .

What is the relationship between PPIH expression and immune cell infiltration in the tumor microenvironment?

Analysis of PPIH expression and immune infiltration has revealed complex relationships in the tumor microenvironment:

• In cholangiocarcinoma, CIBERSORT analysis showed that high PPIH expression correlates with:

  • Decreased naive B cells (19.58% reduction, p < 0.001)

  • Decreased M2 macrophages (35.06% reduction, p < 0.001)

  • Increased follicular helper T cells (77.45% increase, p < 0.001)

  • Increased resting NK cells (33.40% increase, p < 0.01)

  • Increased M0 macrophages (102.57% increase, p < 0.01)

• Stratification by median PPIH expression revealed significant differences in:

  • Naive B cells (p < 0.001)

  • Resting CD4 memory T cells (p < 0.001)

  • M1 macrophages (p < 0.05)

These findings suggest that PPIH may influence anti-tumor immunity through modulation of immune cell populations, particularly affecting T helper cell levels and macrophage polarization .

How does PPIH interact with TP53 mutations in cancer progression?

Research has identified a significant positive correlation between PPIH expression and TP53 mutations in cholangiocarcinoma . This relationship suggests several potential mechanisms:

• PPIH may influence TP53-dependent cellular pathways, affecting tumor suppression mechanisms

• Mutated TP53 might alter cellular splicing machinery, leading to increased PPIH expression

• Both PPIH overexpression and TP53 mutations might represent independent but cooperative oncogenic events

This interaction requires further investigation but presents a promising avenue for understanding the molecular underpinnings of tumor progression and potential therapeutic targeting strategies.

What methodological approaches are most effective for studying PPIH's role in spliceosome function?

Given PPIH's involvement in spliceosome assembly and function, researchers investigating this aspect should consider:

• RNA-seq analysis to identify alternative splicing events associated with PPIH expression levels

• Co-immunoprecipitation studies to identify PPIH interaction partners within the spliceosome complex

• CRISPR-Cas9 gene editing to generate PPIH knockout or knockdown models for functional studies

• RNA splicing reporter assays to directly assess the impact of PPIH modulation on splicing efficiency

• Proteomic analysis of spliceosome components following PPIH perturbation

These approaches can provide complementary insights into how PPIH contributes to normal and aberrant splicing mechanisms in both physiological and pathological contexts.

How can researchers differentiate between the direct molecular effects of PPIH and secondary consequences of its altered expression?

This is a methodologically challenging question that requires careful experimental design:

• Temporal expression studies: Analyzing the time course of molecular changes following PPIH modulation can help distinguish primary from secondary effects

• Dose-response relationships: Titrating PPIH expression levels can reveal threshold-dependent effects

• Rescue experiments: Reintroducing wild-type PPIH in knockout models should reverse direct effects

• Domain-specific mutations: Creating PPIH variants with alterations in specific functional domains can isolate particular activities of the protein

• Single-cell analyses: Examining cell-to-cell variability in PPIH expression and associated phenotypes can reveal cellular contexts that influence PPIH function

Researchers should implement multiple complementary approaches to triangulate direct PPIH functions from secondary consequences of its expression changes.

How does PPIH antibody performance compare across different research applications?

Based on validated research applications, PPIH antibodies demonstrate varying performance characteristics:

Researchers should validate each antibody for their specific application, as performance can vary between manufacturers and even between lots from the same source.

What are the key technical considerations when comparing PPIH with other cyclophilin family members?

The cyclophilin family includes several structurally related proteins that may cross-react with PPIH antibodies. Key considerations include:

• Sequence homology: PPIH shares significant sequence similarity with other cyclophilins, particularly in the PPIase domain

• Subcellular localization: Unlike mitochondrial cyclophilins (e.g., PPIF) , PPIH is primarily associated with the spliceosome

• Functional overlap: Several cyclophilins possess PPIase activity but differ in their protein interaction networks

• Antibody validation: Extensive validation against other cyclophilin family members is essential to ensure specificity

When studying PPIH, researchers should implement controls that can distinguish between PPIH and other cyclophilin family members, particularly in tissues where multiple cyclophilins are expressed.

What methodological approaches can resolve contradictory findings about PPIH function in different studies?

When faced with contradictory findings regarding PPIH function, researchers should consider:

• Cell/tissue context specificity: PPIH may have different functions in different cellular environments

• Experimental approach differences: Varying methodologies may access different aspects of PPIH biology

• Antibody specificity issues: Ensuring antibodies specifically detect PPIH and not related family members

• Expression level considerations: Natural versus forced overexpression may yield different results

• Temporal dynamics: Acute versus chronic alterations in PPIH levels may have opposing effects

A systematic meta-analysis approach that accounts for these variables can help resolve seemingly contradictory findings and develop a more nuanced understanding of PPIH function.

What are the most promising avenues for therapeutic targeting of PPIH in cancer?

Based on current understanding of PPIH biology, several therapeutic strategies warrant investigation:

• Small molecule inhibitors of PPIH PPIase activity

• Disruption of PPIH interactions with spliceosome components

• Combination approaches targeting both PPIH and TP53 pathways given their correlation

• Immunomodulatory strategies addressing PPIH-associated alterations in immune cell infiltration

• RNA-based approaches to modulate PPIH expression levels in tumor cells

Researchers pursuing these approaches should consider both direct anti-tumor effects and potential impacts on the tumor microenvironment, particularly immune cell function.

What novel technological approaches might advance our understanding of PPIH biology?

Emerging technologies that could significantly advance PPIH research include:

• Single-cell multi-omics: Integrating transcriptomic, proteomic, and epigenomic data at single-cell resolution

• Spatial transcriptomics: Mapping PPIH expression and associated molecular changes within the tissue architecture

• Cryo-electron microscopy: Resolving the structural interactions between PPIH and spliceosome components

• CRISPR screening: Identifying synthetic lethal interactions with PPIH to uncover vulnerabilities in PPIH-overexpressing tumors

• Patient-derived organoids: Testing PPIH-targeted interventions in more physiologically relevant models

These approaches could provide unprecedented insights into PPIH function and its therapeutic potential in various disease contexts.

How might comparative studies across cancer types improve our understanding of PPIH as a biomarker?

Cross-cancer type analysis of PPIH expression and function could:

• Identify common versus cancer-specific aspects of PPIH biology

• Establish whether PPIH-associated immune infiltration patterns are conserved across tumor types

• Determine whether the prognostic significance of PPIH varies by cancer type or subtype

• Reveal potential cancer-specific interaction partners that modulate PPIH function

• Guide the development of pan-cancer versus cancer-specific PPIH-targeted therapeutic strategies

Such comparative analyses would benefit from standardized methodologies for PPIH detection and quantification to ensure comparable results across studies and cancer types.