PAIP1 Antibody

What is PAIP1 and what are its primary functions in cellular processes?

PAIP1 functions as a translation regulator that interacts with poly(A) tail binding protein (PABP) and the translation initiation factor eIF4G to facilitate translation initiation . It stimulates translation by inducing the circularization of mRNA through its interaction with eIF3G and eIF4A . Additionally, PAIP1 inhibits degradation of mRNA by participating in inhibiting deadenylation .

Structurally, PAIP1 contains two distinct PABP-binding motifs (PAMs): PAM1 binds to RNA recognition motif 2 in the N-terminus of PABP, while PAM2 binds to the PABC domain of PABP . The gene is located at the 5p12 chromosomal location .

What applications are validated for PAIP1 antibodies in research?

Based on published literature and manufacturer data, PAIP1 antibodies have been validated for multiple applications:

Recommended dilutions for common applications include WB (1:500-1:2000), IP (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate), and IF/ICC (1:200-1:800) .

What are the typical molecular weights observed for PAIP1 in Western blot analysis?

While the calculated molecular weight of PAIP1 is 70 kDa, observed molecular weights in Western blot analysis include bands at 60 kDa, 51 kDa, and 40 kDa . This discrepancy could be due to post-translational modifications, proteolytic processing, or alternative splicing variants. When troubleshooting Western blots, researchers should be aware of these potential band variations to correctly identify PAIP1.

How does PAIP1 expression correlate with cancer progression and what experimental approaches best demonstrate this relationship?

PAIP1 overexpression has been documented in multiple cancer types, including oral squamous cell carcinoma (OSCC), liver cancer, breast cancer, and others . In OSCC, high PAIP1 expression correlates with advanced stage, lymph node metastasis (LNM), and worse pattern of invasion .

To investigate this correlation, researchers should consider a multi-platform experimental approach:

• Gene expression analysis: Utilize public databases such as GEO, TCGA, and CPTAC to analyze PAIP1 mRNA and protein levels across cancer types

• IHC scoring: Develop a standardized scoring system for PAIP1 expression in patient samples and correlate with clinical parameters

• In vitro functional assays: Perform colony formation assays, invasion assays, and evaluate MMP9 activity after PAIP1 knockdown

• Correlation analysis: Assess relationships between PAIP1 expression and other established cancer biomarkers (e.g., correlation between PAIP1 and pSRC in OSCC)

In breast cancer studies, PAIP1 expression was found to be positively correlated with cyclin E2, providing insight into its mechanism of action in promoting cell proliferation .

What are the mechanistic pathways through which PAIP1 knockdown affects cancer cell behavior?

PAIP1 knockdown has been shown to inhibit cancer cell proliferation, invasion, and migration through several mechanisms:

A comprehensive experimental approach should include RNA-seq analysis to identify differentially expressed genes after PAIP1 knockdown, followed by pathway enrichment analysis and validation of key targets by qPCR and Western blot .

How can researchers effectively assess PAIP1 protein stability regulation in experimental systems?

PAIP1 protein is regulated through the ubiquitin-proteasome pathway. Key considerations for studying this regulation include:

• Proteasome inhibitor experiments: Treatment with MG132 or lactacystin significantly increases endogenous PAIP1 protein levels in HEK293T and HeLa cells, indicating proteasome-dependent regulation

• Ubiquitin ligase identification: Among Nedd4 family ubiquitin ligases, WWP2 specifically downregulates PAIP1 protein levels by targeting it for ubiquitination and proteasomal degradation

• Mapping interaction domains: The interaction between WWP2 and PAIP1 occurs through the WW domain of WWP2 and the PABP-binding motif 2 (PAM2) of PAIP1. The two consecutive PXXY motifs in PAM2 are required for WWP2-mediated ubiquitination and degradation

• Functional assessment: Ectopic expression of WWP2 decreases translational stimulatory activity with the degradation of PAIP1

When designing experiments to study PAIP1 stability, researchers should include appropriate controls, such as PABP and Paip2, which are not affected by proteasome inhibitors .

What considerations should be made when studying PAIP1's role in translation termination and readthrough?

PAIP1 has been shown to influence translation termination at premature termination codons (PTCs) through its interaction with PABP. Key experimental considerations include:

• Cell-free translation systems: Use luciferase reporter constructs containing PTCs to directly measure readthrough efficiency

• Poly(A) tail requirements: Include mRNA variants with and without poly(A) tails, as the poly(A) tail is required for PABP and PAIP activities in relation to the PTC

• Protein titration: Test different concentrations of recombinant proteins (PAIP1, PAIP2, PABP) to determine dose-dependent effects on readthrough

• Competition assays: Analyze the effects of simultaneous addition of PABP and PAIP1/PAIP2 to understand competitive binding interactions

The experimental data indicates that PAIP1 and PAIP2 increase the readthrough of PTCs by controlling PABP activity during translation termination, with readthrough increasing 1.5- and 1.8-fold respectively compared to control conditions .

How does loss of PAIP1 affect cellular stress responses and what methodologies best capture these effects?

Loss of PAIP1 leads to activation of cellular stress response pathways with significant implications for cell survival. Studies in Drosophila have shown that:

• Translation reduction: PAIP1 depletion causes reduced protein translation

• Proteotoxic stress: Loss of PAIP1 leads to increased proteotoxic stress

• Integrated stress response (ISR) activation: PAIP1 depletion promotes phosphorylation of eIF2α via the kinase PERK

• Transcription factor regulation: Loss of PAIP1 upregulates the transcription factor gene Xrp1, which contributes to apoptotic cell death and eIF2α phosphorylation

• Translational control: PAIP1 depletion increases Xrp1 translation mediated by its 5'UTR

To capture these effects, researchers should employ multiple complementary approaches:

• Western blot analysis for detecting phosphorylated eIF2α and other ISR markers

• Polysome profiling to assess global translation efficiency

• 5'UTR reporter assays to evaluate translational control

• Apoptosis assays to quantify cell death

• Genetic rescue experiments with ISR pathway components

What methodological considerations are important when analyzing PAIP1 expression in clinical cancer samples?

When analyzing PAIP1 expression in clinical cancer samples, researchers should consider:

• Multi-omics approach: Integrate data from multiple platforms (transcriptomics, proteomics) as done with GEO, TCGA, and CPTAC datasets for OSCC

• Correlation with clinical parameters: Analyze PAIP1 expression in relation to cancer stage, lymph node metastasis, pattern of invasion, and patient survival

• Matched normal-tumor comparison: Compare PAIP1 expression in cancer samples with matched normal tissues to establish baseline differences

• Validation across cohorts: Confirm findings in independent patient cohorts to ensure reproducibility

• Co-expression analysis: Investigate correlations between PAIP1 and other markers, such as the positive correlation between PAIP1 and cyclin E2 in breast cancer tissues or PAIP1 and pSRC in OSCC

For IHC analysis of PAIP1 in tissue samples, validated antibodies with appropriate controls should be used, and standardized scoring systems should be implemented to ensure consistent evaluation across samples.

How can researchers effectively validate PAIP1 knockdown efficiency in experimental systems?

Thorough validation of PAIP1 knockdown efficiency is critical for interpreting functional studies. Based on published methodologies:

• Multiple siRNAs: Design and test multiple siRNA sequences targeting different regions of PAIP1 to identify the most effective knockdown

• qPCR validation: Quantify PAIP1 mRNA levels using qPCR with appropriate reference genes (e.g., GAPDH)

• Western blot confirmation: Verify protein knockdown using validated PAIP1 antibodies

• Time course analysis: Assess knockdown efficiency at multiple time points to determine optimal experimental windows

• Functional validation: Confirm biological effects through established PAIP1-dependent phenotypes, such as reduced cell viability

In the HepG2 cell model, researchers validated PAIP1 knockdown using three different siRNAs and confirmed both mRNA reduction (via qPCR) and protein reduction (via Western blot) before proceeding with RNA-seq and functional studies .