PAK1IP1 Antibody

What is PAK1IP1 and what is its biological significance in cancer research?

PAK1IP1 is a protein that interacts with PAK1 (p21-activated kinase 1) and has been identified as having significant implications in cancer biology. Recent research has established that PAK1IP1 is highly expressed in hepatocellular carcinoma (HCC) tumor tissue samples compared to normal tissues . The protein has been characterized as an oncogenic driver in HCC, with expression levels correlating with various clinical features including patient age, gender, tumor stage, and grade .

From a mechanistic perspective, PAK1IP1 appears to play a regulatory role in pyroptosis, a form of programmed cell death. Knockdown of PAK1IP1 has been demonstrated to induce CASP-3-dependent pyroptosis in HCC cells, which subsequently inhibits cancer cell proliferation and migration . This mechanism provides a potential therapeutic avenue for HCC treatment and underscores the importance of PAK1IP1 as a research target.

What types of PAK1IP1 antibodies are currently available for research applications?

Several types of PAK1IP1 antibodies are available for research purposes, each with specific characteristics suited for different experimental applications:

When selecting an antibody, researchers should consider the specific experimental requirements, including target species, application method, and the region of PAK1IP1 being investigated . The monoclonal antibody ABIN2336030, for instance, targets the internal region of PAK1IP1 and has been purified by ammonium sulfate precipitation .

What are the optimal storage and handling conditions for PAK1IP1 antibodies?

PAK1IP1 antibodies, such as ABIN2336030, are typically supplied as liquid formulations in PBS buffer (pH 7.2) containing preservatives like sodium azide (0.09%) . For optimal maintenance of antibody activity and specificity:

• Store antibodies at -20°C unless otherwise specified by the manufacturer

• Avoid repeated freeze-thaw cycles, which can lead to protein denaturation and loss of activity

• When handling, remember that some preservatives like sodium azide are hazardous and should be managed accordingly by trained personnel only

• Aliquot antibodies upon receipt to minimize freeze-thaw cycles

• Allow antibodies to equilibrate to room temperature before opening the vial to prevent condensation

The longevity and performance of PAK1IP1 antibodies depend significantly on proper storage and handling practices. Researchers should always refer to the specific manufacturer's guidelines for the particular antibody being used.

How does PAK1IP1 expression correlate with clinical features in hepatocellular carcinoma?

Comprehensive analysis of PAK1IP1 expression in relation to clinical features of HCC patients has revealed several significant correlations:

These correlations suggest that PAK1IP1 could serve as a predictive biomarker for HCC progression and potentially inform clinical decision-making. Additionally, qRT-PCR analysis has demonstrated that HCC cell lines (particularly Hep3B and HepG2) exhibit higher PAK1IP1 expression compared to normal liver cells . These findings collectively support the oncogenic role of PAK1IP1 in HCC development and progression.

What is the relationship between PAK1IP1 and immune cell infiltration in the tumor microenvironment?

Analysis of immune cell infiltration patterns in relation to PAK1IP1 expression has yielded important insights:

• Most immune cells show downregulation in samples with low PAK1IP1 expression

• Myeloid dendritic cells (mDCs) demonstrate the highest infiltration percentage in tumor samples with high PAK1IP1 expression

• This association suggests PAK1IP1 may influence the immune microenvironment in HCC

The relationship between PAK1IP1 and mDCs infiltration presents potential therapeutic implications. For instance, the tandem action of CD39 and CD73 ectonucleotidases expressed on mDCs can convert ATP to adenosine, an important mediator of immunosuppression in the tumor microenvironment . Targeting these enzymes could potentially reduce the immunosuppressive effects of mDCs and improve treatment outcomes in HCC patients.

Furthermore, myeloid cell receptor tyrosine kinases (RTKs) including TYRO3, AXL, and MERTK have been shown to suppress immune responses. Inhibition of these receptors could potentially reduce the immune suppression function of mDCs, increase CD8+ T cell infiltration, and enhance treatment efficacy .

How does PAK1IP1 regulate pyroptosis in hepatocellular carcinoma cells?

Research has revealed a novel mechanism whereby PAK1IP1 regulates pyroptosis in HCC cells:

• A physical interaction network has been constructed linking PAK1IP1 and 33 pyroptosis-related genes

• Knockdown of PAK1IP1 significantly increased IL-1β levels following lipopolysaccharide (LPS) treatment, indicating enhanced pyroptosis

• Western blot analysis demonstrated that PAK1IP1 inhibition significantly increased protein levels of:

  • CASP-3 (caspase-3)

  • GSDME-N (gasdermin E N-terminal fragment)

  • Cleaved caspase-1

  • GSDMD-N (gasdermin D N-terminal fragment)

These findings indicate that PAK1IP1 knockdown induces CASP-3-dependent pyroptosis in HCC cells. The mechanism was further validated by treating cells with Z-DEVD-FMK, a CASP-3 inhibitor, which partially reversed the anti-proliferative effects of PAK1IP1 knockdown . This suggests that PAK1IP1 may regulate pyroptosis through a CASP-3-dependent mechanism, offering a potential therapeutic strategy for HCC treatment.

What are the optimal protocols for using PAK1IP1 antibodies in Western blotting applications?

For effective Western blotting with PAK1IP1 antibodies, researchers should follow these methodological guidelines:

• Sample preparation:

  • Extract total protein from cells or tissues using RIPA buffer with protease inhibitors

  • Quantify protein concentration using BCA or Bradford assay

  • Denature samples at 95°C for 5 minutes in loading buffer containing SDS and β-mercaptoethanol

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal separation of PAK1IP1 (approximately 55 kDa)

  • Transfer proteins to PVDF or nitrocellulose membranes at 100V for 60-90 minutes in cold transfer buffer

• Antibody incubation:

  • Block membranes with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Incubate with primary PAK1IP1 antibody (typical dilution 1:1000, but optimize based on specific antibody) overnight at 4°C

  • Wash membranes 3-5 times with TBST

  • Incubate with appropriate secondary antibody (based on primary antibody host species) for 1 hour at room temperature

  • Wash thoroughly before detection

• Controls and validation:

  • Include positive control samples (HCC cell lines like Hep3B or HepG2 show high PAK1IP1 expression)

  • Use PAK1IP1 knockdown samples as negative controls where possible

  • Include loading controls (e.g., GAPDH, β-actin) to normalize expression levels

Optimization of antibody concentration is critical, as demonstrated in studies where PAK1IP1 knockdown efficacy was validated by Western blotting . The specificity of the signal can be confirmed by comparing expression levels in normal liver cells versus HCC cell lines.

How can PAK1IP1 antibodies be effectively utilized in ELISA assays for pyroptosis research?

ELISA assays using PAK1IP1 antibodies have been instrumental in studying pyroptosis in HCC. Based on published methodologies:

• Sample preparation:

  • Collect cell culture supernatants or tissue homogenates

  • Centrifuge samples to remove cellular debris

  • Store samples at -80°C or analyze immediately

• ELISA procedure:

  • For detecting pyroptosis markers like IL-1β:

    • Use commercial ELISA kits or develop custom assays using PAK1IP1 antibodies

    • Follow manufacturer's protocol for commercial kits

    • For custom assays, coat plates with capture antibody overnight at 4°C

• Experimental design considerations:

  • Include appropriate treatment groups:

    • Control (untreated) cells

    • LPS-treated cells (to induce pyroptosis)

    • PAK1IP1 knockdown cells

    • Combination of PAK1IP1 knockdown and LPS treatment

• Data analysis:

  • Construct standard curves using recombinant proteins

  • Normalize data to cell number or protein concentration

  • Perform statistical analysis to determine significance of differences between treatment groups

Research has demonstrated that IL-1β levels significantly increase following LPS treatment and PAK1IP1 knockdown in HCC cells, indicating enhanced pyroptosis . This methodology has been crucial in establishing the relationship between PAK1IP1 and pyroptosis regulation in HCC.

What experimental approaches are recommended for studying PAK1IP1 knockdown effects on cell proliferation and migration?

To effectively study PAK1IP1 knockdown effects on cancer cell phenotypes, researchers should consider these methodological approaches:

• PAK1IP1 knockdown strategies:

  • siRNA transfection: Two effective siRNAs (si-PAK1IP1#1 and si-PAK1IP1#2) have been validated for efficient PAK1IP1 knockdown in HCC cells

  • Include appropriate negative controls (non-targeting siRNA)

  • Validate knockdown efficiency using qRT-PCR and Western blotting

• Cell proliferation assays:

  • CCK-8 or MTT assays can be used to measure proliferation rates

  • Plate cells at appropriate density (typically 2-5 × 10³ cells/well in 96-well plates)

  • Measure proliferation at multiple time points (24h, 48h, 72h)

  • Include treatment groups with CASP-3 inhibitor (Z-DEVD-FMK) to investigate pyroptosis mechanisms

• Migration and invasion assays:

  • Transwell assays are effective for assessing migration and invasion capabilities

  • For invasion assays, coat transwell inserts with Matrigel

  • Seed cells in serum-free medium in the upper chamber

  • Use medium containing serum as a chemoattractant in the lower chamber

  • Incubate for 24-48 hours before fixing, staining, and counting migrated/invaded cells

• Image analysis:

  • Capture images at consistent magnification (e.g., scale: 50 μm as used in published studies)

  • Use image analysis software to quantify results

  • Perform blinded counting to avoid bias

Published research has demonstrated that PAK1IP1 knockdown significantly reduces HCC cell proliferation, while treatment with Z-DEVD-FMK increases cell proliferation, suggesting a CASP-3-dependent mechanism . Similarly, Transwell assays have shown that PAK1IP1 knockdown suppresses liver cancer cell invasion and migration after LPS treatment .

How can researchers address inconsistent results when studying PAK1IP1 expression across different HCC samples?

Inconsistencies in PAK1IP1 expression data can arise from several sources. To address these challenges:

• Standardize sample collection and processing:

  • Use consistent protocols for tissue collection and preservation

  • Document clinical parameters including patient age, gender, tumor stage, and grade

  • Consider TP53 mutation status, as PAK1IP1 expression correlates with TP53 mutations

• Account for heterogeneity in expression:

  • Analyze subgroups based on clinical features

  • Consider analyzing multiple regions within tumor samples

  • Use larger sample sizes to account for biological variation

• Employ multiple detection methods:

  • Combine techniques (qRT-PCR, Western blotting, immunohistochemistry)

  • Use validated antibodies that target specific regions of PAK1IP1

  • Include appropriate positive controls (HCC cell lines) and negative controls

• Data normalization strategies:

  • For qRT-PCR: Use multiple reference genes stable in HCC tissues

  • For Western blotting: Normalize to multiple loading controls

  • For database analyses: Review normalization methods used in databases like UALCAN

Published research has shown variable PAK1IP1 expression patterns correlated with different clinical features, emphasizing the importance of stratifying analyses by these parameters . When inconsistencies occur, researchers should consider whether they reflect true biological variation or technical limitations.

What factors should be considered when analyzing the relationship between PAK1IP1 and immune cell infiltration?

The analysis of PAK1IP1's relationship with immune cell infiltration requires careful consideration of several factors:

• Immune cell identification and quantification:

  • Use multiple markers to identify specific immune cell populations

  • Consider both the percentage and absolute numbers of infiltrating cells

  • Apply consistent gating strategies for flow cytometry analyses

• Spatial distribution analysis:

  • Assess the location of immune cells (tumor core vs. periphery)

  • Consider cell-cell interactions between tumor cells and immune cells

  • Use multiplexed immunofluorescence to visualize spatial relationships

• Functional assessment:

  • Measure cytokine production by infiltrating immune cells

  • Assess activation status of immune cells

  • Investigate the suppressive mechanisms in the tumor microenvironment

• Correlation analyses:

  • Control for confounding factors (tumor stage, grade, treatment history)

  • Use appropriate statistical methods for correlation analyses

  • Consider multivariate analysis to account for multiple variables

Research has shown that myeloid dendritic cells have the highest infiltration percentage in tumor samples with high PAK1IP1 expression . The immunosuppressive functions of these cells, including the conversion of ATP to adenosine via CD39 and CD73 ectonucleotidases, should be considered when interpreting these relationships .

How should researchers interpret conflicting data on the mechanisms of PAK1IP1-mediated pyroptosis?

When faced with conflicting data regarding PAK1IP1-mediated pyroptosis mechanisms:

• Evaluate experimental differences:

  • Cell lines used (different HCC cell lines may show variable responses)

  • Knockdown efficiency (partial vs. complete PAK1IP1 knockdown)

  • Experimental conditions (culture conditions, treatment durations)

• Assess pyroptosis detection methods:

  • Multiple markers should be examined (IL-1β, GSDMD-N, GSDME-N, cleaved caspase-1, CASP-3)

  • Combine protein expression analysis with functional assays

  • Consider time-course experiments to capture dynamics of pyroptosis activation

• Validate with mechanistic interventions:

  • Use specific inhibitors (e.g., Z-DEVD-FMK for CASP-3 inhibition)

  • Apply genetic approaches (knockdown/knockout of pathway components)

  • Perform rescue experiments to confirm specificity

• Integrate with other cell death pathways:

  • Consider cross-talk between pyroptosis and apoptosis

  • Evaluate potential compensatory mechanisms

  • Assess concurrent activation of multiple cell death pathways

Current research suggests that PAK1IP1 knockdown induces CASP-3-dependent pyroptosis, as evidenced by increased protein levels of CASP-3, GSDME-N, cleaved caspase-1, and GSDMD-N . The partial reversal of anti-proliferative effects by Z-DEVD-FMK supports this mechanism . When confronting conflicting data, researchers should systematically evaluate these experimental variables and consider the possibility of context-dependent mechanisms.