PTTG2 Antibody, HRP conjugated

What is PTTG2 and what cellular functions does it regulate?

PTTG2 is an intronless homolog of PTTG1 (human pituitary tumor-transforming gene 1) that plays distinct roles in cellular processes. Unlike PTTG1, which functions as a securin preventing premature chromatid separation through interaction with separase, PTTG2 has been shown to lack the ability to interact with separase under experimental conditions . This suggests PTTG2 likely does not function in preventing premature chromatid separation through the same mechanism as PTTG1.

PTTG2 appears to regulate several important cellular processes:

• Cell proliferation and viability (demonstrated in multiple cell types)

• Cell invasion and migration capabilities

• Regulation of apoptosis pathways (potentially through caspase-3 dependent signaling)

• Cellular adhesion and cytoskeletal organization

Studies have shown PTTG2 expression levels are relatively low in various normal human tissues compared to PTTG1, which has complicated research into its functions .

How does PTTG2 expression differ between normal and pathological tissues?

PTTG2 expression varies significantly between normal and pathological tissues:

• In psoriasis: PTTG2 is significantly overexpressed in psoriatic epidermis cells compared to normal cells, showing approximately three times higher mRNA levels and correspondingly elevated protein levels .

• In glioblastoma: Research indicates PTTG2 is overexpressed in glioblastoma cells, contributing to the aggressive nature of this malignancy .

• In normal tissues: PTTG2 generally exhibits low expression levels across various healthy human tissues, as demonstrated through cDNA array analyses .

This differential expression pattern makes PTTG2 a potential biomarker for certain pathological conditions and possibly a therapeutic target.

What are the functional differences between PTTG1 and PTTG2?

Despite their high sequence homology, PTTG1 and PTTG2 demonstrate important functional differences:

These biochemical differences suggest PTTG2 participates in biological responses distinct from PTTG1, despite their structural similarities. Researchers investigating either protein should be aware of these differences when designing experiments and interpreting results.

What experimental approaches are recommended for PTTG2 protein detection using HRP-conjugated antibodies?

When detecting PTTG2 protein using HRP-conjugated antibodies, consider these methodological approaches:

Western Blotting Protocol Optimization:

• Use multiple PTTG2 antibodies raised against different epitopes to confirm specificity

• Include positive controls with known PTTG2 expression

• Implement longer exposure times due to the typically low PTTG2 expression levels

• Consider enhanced chemiluminescence (ECL) detection systems for improved sensitivity

Based on published research, scientists have successfully used antibodies raised against GST-tagged PTTG2, His-tagged PTTG2, and synthetic peptides located at the C-terminal region . The low expression levels make detection challenging, requiring careful optimization of detection methods.

How can researchers effectively silence PTTG2 expression for functional studies?

Silencing PTTG2 expression presents unique challenges due to its sequence similarity with PTTG1. Based on published research:

• shRNA approach challenges:
  Multiple shRNA lentiviral clones targeting different regions within the PTTG2 ORF have been tested, but achieving high specificity without affecting PTTG1 has proven difficult . For example, researchers tested five different shRNA lentiviral clones and found none could specifically silence PTTG2 without altering PTTG1 mRNA levels.

• Optimal approach based on published data:
  The most effective strategy identified used a shRNA containing two mismatches compared to PTTG1, which reduced PTTG2 mRNA levels by 62% but also reduced PTTG1 by 65% . This requires careful experimental design to distinguish PTTG2-specific effects.

• Validation requirements:

  • Confirm knockdown efficiency at both mRNA and protein levels

  • Include controls for PTTG1 expression

  • Consider rescue experiments with PTTG1 cDNA to differentiate phenotypes

Due to these challenges, researchers should incorporate thorough validation steps when conducting PTTG2 silencing experiments.

How can PTTG2 antibodies be utilized in glioblastoma research?

PTTG2 antibodies offer several valuable applications in glioblastoma research:

Monitoring PTTG2 expression patterns:
Studies have demonstrated that PTTG2 overexpression promotes cell proliferation and invasion during glioblastoma progression . HRP-conjugated PTTG2 antibodies can be used to:

• Quantify PTTG2 expression levels in patient samples

• Compare expression between different glioblastoma grades

• Correlate PTTG2 levels with patient prognosis and survival outcomes

Investigating molecular mechanisms:
Research indicates PTTG2 overexpression inhibits cell apoptosis in glioblastoma by affecting caspase-3-dependent signaling pathways . PTTG2 antibodies can help:

• Track changes in PTTG2 expression following therapeutic interventions

• Identify protein interactions in signaling cascades

• Evaluate the relationship between PTTG2 and apoptotic markers

Therapeutic target validation:
As PTTG2 has been suggested as a potential therapeutic target for treating glioblastoma , antibodies can be used to:

• Confirm target engagement in drug development

• Monitor changes in PTTG2 expression following experimental treatments

• Develop immunohistochemical protocols for patient stratification

What methodological considerations are important when using PTTG2 antibodies to study cell invasion?

When investigating cell invasion processes, researchers should consider:

Matrigel Transwell assay optimization:
Research has demonstrated PTTG2's role in cell invasion using the Matrigel Transwell assay, with PTTG2 overexpression significantly increasing invasive cell numbers (1.63 times higher) compared to untreated controls . Key considerations include:

• Sample preparation and cell density standardization

• Incubation time optimization (typically 24-48 hours)

• Quantification methods (counting cells from multiple random high-power fields)

Controls and validation:

• Include both PTTG2 overexpression and knockdown conditions

• Use siRNA-PTTG2 groups as negative controls (shown to reduce invasion to 28% of untreated controls)

• Confirm PTTG2 expression levels correlate with observed phenotypic changes

Complementary assays:

• Combine invasion assays with proliferation assessment (e.g., MTT assay)

• Evaluate cytoskeletal changes using α-tubulin immunostaining

• Monitor EMT markers like vimentin and E-cadherin that may be affected by PTTG2

How do PTTG2 expression levels affect cell adhesion and what experimental methods best measure this relationship?

PTTG2 has been demonstrated to play a critical role in cell adhesion:

Experimental approaches:

• Cell aggregation assays: When PTTG2 is silenced, cells show impaired ability to form dense spheroidal aggregates, appearing more dispersed and forming only loose clumps when grown on poly-HEMA plates . This indicates compromised cell-cell interconnection.

• Cytoskeletal analysis: PTTG2-depleted cells show altered patterns of α-tubulin compared to control cells, suggesting that reduced intercellular adhesions are coupled with abnormal distribution of cytoskeletal proteins .

• Apoptosis assessment: Loss of adhesion in PTTG2-depleted cells correlates with increased cell death (20-30%), measured by the number of cells presenting fragmented DNA (subG1 peak) .

Methodological considerations:

• Use poly-HEMA-coated plates to assess cell-cell adhesion in suspension

• Include methylcellulose in culture medium as a control to minimize cell-cell contacts

• Monitor both structural changes and cell survival metrics

• Examine levels of acetylated α-tubulin, which are elevated in PTTG2-depleted cells

The relationship between PTTG2 and cell adhesion appears specific, as PTTG1-silenced cells maintain normal cell-cell adhesion properties, forming dense typical round colonies with tightly associated cell-cell contacts .

How can researchers validate PTTG2 antibody specificity, particularly in distinguishing between PTTG1 and PTTG2?

Ensuring PTTG2 antibody specificity is crucial due to the high sequence homology with PTTG1. Recommended validation approaches include:

Multiple antibody comparison:
Researchers have successfully used three different PTTG2 antibodies raised against different targets:

• GST-tagged PTTG2

• His-tagged PTTG2

• Synthetic peptide located at the C-terminal region of PTTG2

Genetic controls:

• Use PTTG1 knockout models (e.g., HCT116 pttg1−/− cells) to confirm antibody specificity

• Perform experiments in cell lines with PTTG1 silencing to ensure PTTG2 detection is not affected

• Include overexpression controls with tagged versions of both proteins

Cross-reactivity testing:

• Perform immunoprecipitation with PTTG1 and PTTG2 separately

• Run Western blots with recombinant PTTG1 and PTTG2 proteins

• Consider peptide competition assays using specific peptides for each protein

What are common technical challenges when using PTTG2 antibodies in experimental systems?

Researchers face several technical challenges when working with PTTG2 antibodies:

Low expression levels:
PTTG2 typically shows significantly lower expression compared to PTTG1, making detection challenging . This is not due to antibody sensitivity issues, as multiple antibodies show similar results. Strategies to address this include:

• Using more sensitive detection methods

• Increasing protein loading amounts for Western blots

• Employing signal amplification techniques

Specificity concerns:
Due to high sequence homology between PTTG family members, ensuring antibody specificity is crucial. Researchers should:

• Use antibodies targeting unique epitopes

• Include appropriate positive and negative controls

• Validate results using complementary detection methods

Variability across tissue types:
PTTG2 expression varies across different tissues and cell types, requiring optimization for each experimental system. Expression has been studied in:

• Human embryonic kidney HEK293T cells

• hTERT-immortalized retinal pigment epithelial cell line RPE1

• Human breast adenocarcinoma MCF7 cells

• Cervical cancer cell line HeLa

How can PTTG2 antibodies contribute to psoriasis research?

PTTG2 has been implicated in psoriasis pathogenesis, with significantly higher expression levels in psoriatic cells than in normal cells . PTTG2 antibodies can advance psoriasis research through:

Expression profiling:

• Quantify PTTG2 levels in patient samples vs. healthy controls

• Compare expression across different psoriasis subtypes and severities

• Monitor changes in response to treatments

Functional investigation:
Research has shown that knockdown of PTTG2 inhibits the viability and migration of HaCaT cells (a keratinocyte cell line) . PTTG2 antibodies can help:

• Track PTTG2 localization in affected tissues

• Examine co-localization with other psoriasis-associated proteins

• Identify potential protein interactions in psoriatic lesions

Biomarker development:

• Assess correlation between PTTG2 levels and disease activity

• Evaluate PTTG2 as a predictive marker for treatment response

• Develop standardized immunohistochemistry protocols for clinical applications

What approaches can be used to investigate PTTG2's role in epithelial-to-mesenchymal transition?

Epithelial-to-mesenchymal transition (EMT) is a critical process in development and disease. PTTG2 silencing has been linked to EMT, and researchers can investigate this connection using:

Marker analysis:
Track changes in key EMT markers when PTTG2 levels are modulated:

• E-cadherin (epithelial marker)

• Vimentin (mesenchymal marker)

• Other markers such as N-cadherin, ZO-1, and SNAIL family transcription factors

Morphological assessment:
PTTG2-depleted cells show altered morphology, including:

• Changes in cell-cell adhesion

• Modified cytoskeletal organization

• Altered α-tubulin distribution

Functional assays:

• Migration assays to assess changes in cell motility

• 3D culture models to examine morphogenesis

• Invasion assays to quantify changes in metastatic potential

Signal pathway analysis:
Determine how PTTG2 interfaces with known EMT-inducing pathways:

• TGF-β signaling

• Wnt/β-catenin pathway

• Notch signaling

This multifaceted approach can help elucidate the mechanisms by which PTTG2 influences the EMT process and its implications for development and disease.