PDLIM2 Antibody, HRP conjugated

What is PDLIM2 and what are its primary functions in cellular biology?

PDLIM2 (PDZ and LIM domain protein 2) functions as a ubiquitin E3 ligase that promotes proteasomal degradation of various transcription factors, particularly NF-κB/RelA and STAT proteins. It serves as an adapter protein located at the actin cytoskeleton that promotes cell attachment and is necessary for epithelial cell migration . PDLIM2 enhances cell adhesion to extracellular matrix components like collagen and fibronectin while suppressing anchorage-independent growth, which is a hallmark of cancer cells . Its most notable role appears to be as a tumor suppressor, particularly in lung cancer, where its expression correlates with therapeutic response and better prognosis .

What experimental applications are suitable for PDLIM2 Antibody, HRP conjugated?

PDLIM2 Antibody, HRP conjugated is primarily suitable for western blotting, immunohistochemistry on paraffin-embedded tissues (IHC-P), and certain ELISA applications . The antibody specifically recognizes human PDLIM2 protein, making it valuable for clinical samples and human cell line-based research . While unconjugated versions of PDLIM2 antibodies can be used for immunocytochemistry/immunofluorescence (ICC/IF) applications, the HRP-conjugated version is better suited for applications requiring enzymatic detection systems rather than fluorescence-based imaging . For optimal results, researchers should validate specific dilution factors for their experimental systems, typically starting with manufacturer recommendations (e.g., 1:1000 for western blotting).

What controls should be included when using PDLIM2 Antibody, HRP conjugated?

When using PDLIM2 Antibody, HRP conjugated, several controls are essential for experimental validation. Positive controls should include cell lines or tissues known to express PDLIM2, such as Jurkat T cells which demonstrate relatively abundant PDLIM2 protein expression . Negative controls should include HTLV-I-transformed T-cell lines, which have been shown to express significantly decreased PDLIM2 levels . Additional technical controls should include: (1) Isotype-matched irrelevant antibody controls to assess non-specific binding; (2) Loading controls like β-actin or HSP90 which are insensitive to PDLIM2 expression changes ; (3) PDLIM2 knockout or knockdown samples, where available, to confirm antibody specificity; and (4) No-primary antibody controls to ensure secondary detection systems aren't producing background signals.

How can PDLIM2 Antibody, HRP conjugated be optimized for detecting low PDLIM2 expression in cancer samples?

Detecting PDLIM2 in cancer samples presents a significant challenge since PDLIM2 is often epigenetically repressed in cancers such as lung cancer . For optimal detection in these contexts, researchers should implement several optimization strategies: (1) Employ extended incubation times (overnight at 4°C) with higher antibody concentrations (1:500 rather than standard 1:1000 dilutions); (2) Utilize enhanced chemiluminescent substrates specifically designed for low-abundance proteins; (3) Incorporate antigen retrieval methods for FFPE samples, particularly citrate buffer (pH 6.0) with heat-induced epitope retrieval; (4) Consider protein enrichment via immunoprecipitation before western blotting when working with clinical samples; and (5) Use signal amplification systems such as tyramide signal amplification (TSA) to enhance sensitivity while maintaining specificity. Additionally, preprocessing samples with epigenetic drugs like HDAC inhibitors in ex vivo cultures can temporarily upregulate PDLIM2 expression, facilitating better detection .

What methodological considerations should be taken when studying PDLIM2-Tax interactions using this antibody?

When investigating PDLIM2-Tax interactions, researchers should implement a multi-technique approach for comprehensive analysis. First, reciprocal co-immunoprecipitation assays should be performed using both anti-PDLIM2 and anti-Tax antibodies, followed by immunoblotting with the HRP-conjugated PDLIM2 antibody . For subcellular localization studies, cell fractionation is critical—prepare cytoplasmic and nuclear extracts separately as PDLIM2 shuttles Tax to the nucleoplasm for degradation .

For studying the dynamics of interaction, researchers should include proteasome inhibitors (e.g., 10μM MG132) in selected experimental groups to prevent Tax degradation, allowing better visualization of the Tax-PDLIM2 complex . Additionally, protein stability assays using cycloheximide (CHX) chase experiments (10μM CHX) with timed collections will reveal the kinetics of PDLIM2-mediated Tax degradation . For visualizing subcellular interactions, supplement biochemical studies with immunofluorescence co-localization experiments examining PDLIM2, Tax, and proteasome markers simultaneously, particularly focusing on nuclear matrix regions which serve as proteolytic sites distinct from PML nuclear bodies .

How can PDLIM2 Antibody be used to investigate the synergistic relationship between PDLIM2 and PDLIM7?

To investigate the synergistic relationship between PDLIM2 and PDLIM7, researchers should employ multiple complementary approaches using PDLIM2 antibody. Begin with endogenous co-immunoprecipitation experiments using anti-PDLIM7 antibodies in cells expressing both proteins (e.g., RAW264.7 cells), followed by immunoblotting with PDLIM2 antibody, HRP conjugated . To map interaction domains, perform co-transfection experiments with tagged wild-type and deletion mutants of both PDLIM2 and PDLIM7, focusing on PDZ and LIM domains which are likely interaction points .

For functional synergy studies, implement the following experimental design: (1) Transfect NIH3T3 cells with plasmids encoding p65, PDLIM2, and PDLIM7 in various combinations; (2) Perform nuclear fractionation to isolate soluble nuclear p65; and (3) Analyze with immunoblotting to detect changes in p65 levels that reflect synergistic degradation activity . This approach will reveal whether PDLIM2 and PDLIM7 form functional heterodimers that enhance E3 ligase activity compared to individual proteins, similar to other RING finger-type E3 ligase heterodimers like Mdm2-MdmX, BRCA1-BARD1, and RING1B-Bmi1 .

What strategies can resolve contradictory findings in PDLIM2 expression levels across different cancer cell lines?

Contradictory findings regarding PDLIM2 expression across cancer cell lines can be systematically addressed through a comprehensive analytical approach. First, implement multi-level detection methods: quantify both protein (using HRP-conjugated PDLIM2 antibody) and mRNA (using RT-qPCR) levels simultaneously in the same samples to determine whether discrepancies occur at transcriptional or post-translational levels . For RT-qPCR, use validated primers such as: human PDLIM2 forward 5′-GCCCATCATGGTGACTAAGG, reverse 5′-ATGGCCACGATTATGTCTCC .

Second, analyze epigenetic regulation by examining promoter methylation status and histone modifications at the PDLIM2 locus, as PDLIM2 is frequently epigenetically repressed in cancers . Third, assess protein stability through cycloheximide chase experiments comparing PDLIM2 degradation rates across cell lines . Fourth, examine subcellular localization patterns via cellular fractionation and immunofluorescence, as PDLIM2 function depends on proper localization . Finally, consider context-dependent regulatory mechanisms by analyzing the expression and activation status of NF-κB and STAT transcription factors, which may reciprocally regulate PDLIM2 expression. This systematic approach can reconcile seemingly contradictory findings by identifying cell-specific regulatory mechanisms.

How can PDLIM2 Antibody be utilized to evaluate the efficacy of epigenetic drugs in restoring PDLIM2 expression?

PDLIM2 antibody, HRP conjugated, serves as a critical tool for evaluating epigenetic drug efficacy in restoring PDLIM2 expression in cancer models. Researchers should design time-course experiments where cancer cell lines with epigenetically silenced PDLIM2 (such as HTLV-I-transformed T cells or lung cancer cells) are treated with escalating doses of epigenetic modifiers like DNA methyltransferase inhibitors (e.g., 5-azacytidine) or histone deacetylase inhibitors . At each timepoint (typically 24, 48, and 72 hours), collect protein samples for western blotting with the HRP-conjugated PDLIM2 antibody and RNA samples for parallel RT-qPCR assessment.

For translational relevance, ex vivo treatments of patient-derived tumor samples should be performed followed by immunohistochemistry using the PDLIM2 antibody to evaluate restoration of expression in actual tumor tissues . Additionally, chromatin immunoprecipitation (ChIP) assays examining histone modification changes at the PDLIM2 promoter will correlate epigenetic alterations with expression changes. For functional validation, researchers should assess downstream effects on NF-κB/STAT signaling and perform cell transformation assays to determine whether PDLIM2 restoration is sufficient to reverse malignant phenotypes .

What role does PDLIM2 detection play in predicting therapeutic responses to immunotherapy and chemotherapy?

PDLIM2 detection using specific antibodies can serve as a potential predictive biomarker for therapeutic responses to both immunotherapy and chemotherapy. Studies have established that PDLIM2 expression is significantly associated with better therapeutic outcomes in lung cancer patients . For clinical applications, researchers should develop a standardized immunohistochemistry protocol using PDLIM2 antibody on patient tumor biopsies with clearly defined scoring criteria based on staining intensity and percentage of positive cells.

In research settings, implementing a comprehensive biomarker analysis workflow is essential: (1) Perform baseline PDLIM2 immunohistochemistry or western blotting on patient samples prior to treatment initiation; (2) Correlate expression levels with response rates to anti-PD-1/PD-L1 immunotherapy and standard chemotherapy regimens; (3) Monitor dynamic changes in PDLIM2 expression during treatment by analyzing serial biopsies when available; and (4) Integrate PDLIM2 data with other biomarkers including PD-L1 expression, tumor mutational burden, and immune infiltration patterns . Mechanistically, PDLIM2 likely predicts treatment response through its ability to increase expression of genes involved in antigen presentation and T-cell activation while repressing multidrug resistance genes, thereby rendering cancer cells more vulnerable to immune attacks and therapies .

How can PDLIM2 Antibody be used in developing mouse models to study PDLIM2's role in cancer development and therapeutic resistance?

PDLIM2 antibody is instrumental in developing and characterizing mouse models for studying PDLIM2's role in cancer development and therapeutic resistance. When establishing PDLIM2 knockout or lung epithelial-specific deletion mouse models, researchers should use the antibody to confirm deletion efficiency via western blotting and immunohistochemistry of target tissues . For inducible models, the antibody can verify temporal control of PDLIM2 expression following promoter activation.

In experimental workflows, researchers should induce lung cancer in PDLIM2-knockout and wild-type mice using carcinogens or genetic drivers, then implement treatment regimens including chemotherapy, anti-PD-1 immunotherapy, or epigenetic drugs . At defined timepoints, collect tumor tissue for comprehensive analysis including: (1) Immunohistochemistry with PDLIM2 antibody to confirm continued deletion or rescue expression; (2) Western blotting for downstream signaling markers including NF-κB/RelA and STAT3 activation status; (3) Transcriptomic analysis correlating PDLIM2 expression with genes involved in antigen presentation, T-cell activation, and multidrug resistance; and (4) Flow cytometry of tumor-infiltrating lymphocytes to assess immune microenvironment changes . This multi-faceted approach using PDLIM2 antibody as a key analytical tool will establish causative relationships between PDLIM2 expression and therapeutic responses in vivo.

What are the common pitfalls when using PDLIM2 Antibody, HRP conjugated, and how can they be addressed?

Several common pitfalls may arise when working with PDLIM2 Antibody, HRP conjugated. First, non-specific background signals can occur due to excess antibody concentration. This can be addressed by careful titration experiments to determine optimal concentrations (typically starting at 1:1000 and adjusting as needed) and implementing more stringent washing procedures with increased Tween-20 concentration (0.1-0.3%) in TBST buffers .

Second, inconsistent results across experiments often stem from sample degradation during preparation. To prevent this, always include protease inhibitor cocktails in lysis buffers and maintain samples at 4°C throughout processing . Third, false negative results are common when working with cancer samples due to low PDLIM2 expression. Implement signal enhancement strategies such as extended exposure times with high-sensitivity chemiluminescent substrates or consider sample concentration methods .

Fourth, HRP activity degradation can occur with repeated freeze-thaw cycles of the conjugated antibody. Aliquot the antibody upon receipt and store at -20°C, avoiding more than two freeze-thaw cycles . Finally, unexpected bands on western blots may represent PDLIM2 complexes with other proteins like PDLIM7 or ubiquitinated forms of PDLIM2 targets. Confirm band identity through immunoprecipitation followed by mass spectrometry or by comparing band patterns with PDLIM2-overexpressing positive control samples .

How should researchers optimize PDLIM2 protein extraction from different cellular compartments?

Optimal PDLIM2 protein extraction from different cellular compartments requires specialized buffer systems and methodical approaches due to PDLIM2's distribution across cytoskeletal, cytoplasmic, and nuclear fractions. For total cell lysates, use RIPA buffer supplemented with 1% Na-deoxycholate, 1% NP-40, and 1mM DTT alongside protease inhibitor cocktail and 1mM PMSF . For cytoplasmic extraction, employ hypotonic buffer (10mM HEPES pH 7.9, 10mM KCl, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT) with gentle cell lysis using 0.5% NP-40 .

For nuclear extraction, after cytoplasmic removal, treat nuclear pellets with high-salt buffer (20mM HEPES pH 7.9, 400mM NaCl, 1mM EDTA, 1mM EGTA, 1mM DTT) . To specifically isolate nuclear matrix-associated PDLIM2, perform sequential extraction: after high-salt nuclear extraction, treat the insoluble pellet with nuclease (25U/ml DNase I, 5mM MgCl₂) followed by extraction with 0.25M ammonium sulfate . For cytoskeletal fractions, after cytoplasmic and nuclear extraction, solubilize the remaining pellet in buffer containing 1% SDS and 1% Triton X-100.

Critical parameters include maintaining samples at 4°C throughout processing, using gentle mechanical disruption (avoid sonication which can disrupt protein-protein interactions), and immediately processing samples for western blotting with HRP-conjugated PDLIM2 antibody to minimize protein degradation.

What are the key considerations for quantitative analysis of PDLIM2 expression across different experimental conditions?

Quantitative analysis of PDLIM2 expression across different experimental conditions requires rigorous standardization and controls to ensure reliable comparisons. First, establish a dynamic range for detection by performing a standard curve using recombinant PDLIM2 protein at known concentrations or serially diluted positive control lysates (e.g., Jurkat cells) . For western blotting quantification, always include technical replicates (minimum triplicate) and normalize PDLIM2 signal intensity to loading controls that remain stable across experimental conditions (β-actin or HSP90 have been validated for PDLIM2 studies) .

For densitometry analysis, use software that provides background subtraction capabilities and linear range confirmation. When comparing PDLIM2 expression across tissue types or cell lines with vastly different basal expression (e.g., Jurkat T cells versus HTLV-I-transformed T cells), supplement protein analysis with mRNA quantification using validated RT-qPCR primers (human PDLIM2: forward 5′-GCCCATCATGGTGACTAAGG, reverse 5′-ATGGCCACGATTATGTCTCC) .

For temporal studies examining PDLIM2 regulation, standardize collection times to account for potential circadian expression patterns, and implement time-course experiments with multiple sampling points rather than endpoint-only measurements. When monitoring PDLIM2 restoration following treatments, calculate both fold-change from baseline and absolute expression levels relative to positive controls to accurately assess the degree of recovery .

How might PDLIM2 Antibody be used to investigate the relationship between PDLIM2 and tumor immune microenvironment?

PDLIM2 antibody represents a valuable tool for investigating the relationship between PDLIM2 expression and the tumor immune microenvironment. Researchers should implement multiplex immunohistochemistry or immunofluorescence techniques combining PDLIM2 detection with markers for various immune cell populations (CD8+ T cells, regulatory T cells, macrophages, etc.) in tumor tissue sections . This approach allows spatial correlation between PDLIM2 expression in tumor cells and immune infiltration patterns.

Flow cytometry analysis of dissociated tumors can further characterize immune populations in PDLIM2-high versus PDLIM2-low regions. For mechanistic studies, researchers should establish co-culture systems where PDLIM2-expressing or PDLIM2-deficient cancer cells are cultured with immune cells, followed by functional assays measuring immune activation (cytokine production, proliferation, cytotoxicity) . Transcriptional profiling comparing PDLIM2-expressing versus PDLIM2-silenced tumors should focus on genes involved in antigen presentation, T-cell activation, and immune checkpoint pathways .

Additionally, in vivo models using PDLIM2 global or conditional knockout mice should analyze changes in tumor-infiltrating lymphocytes and response to immunotherapies like anti-PD-1 . This comprehensive approach will elucidate how PDLIM2 influences the immune landscape, potentially explaining its role in determining responsiveness to immunotherapy and providing rationale for combination therapeutic strategies.

What experimental designs could elucidate the epigenetic mechanisms responsible for PDLIM2 silencing in cancer?

To elucidate the epigenetic mechanisms responsible for PDLIM2 silencing in cancer, researchers should implement a comprehensive epigenome analysis approach. Begin with bisulfite sequencing of the PDLIM2 promoter region in paired normal and cancer tissues/cell lines to map DNA methylation patterns . Complement this with chromatin immunoprecipitation (ChIP) assays using antibodies against repressive histone marks (H3K27me3, H3K9me3) and activating marks (H3K4me3, H3K27ac) at the PDLIM2 locus .

To identify specific epigenetic regulators responsible for PDLIM2 silencing, implement CRISPR screens targeting chromatin modifiers, followed by PDLIM2 antibody-based western blotting to identify hits that restore expression. For deeper mechanistic understanding, perform ChIP-seq for identified histone modifiers and DNA methyltransferases (DNMTs) across the genome in cancer cells before and after epigenetic drug treatment that restores PDLIM2 expression .

For translational relevance, develop an epigenetic biomarker panel by correlating specific PDLIM2 promoter methylation patterns or histone modifications with protein expression levels (measured by the HRP-conjugated PDLIM2 antibody) and clinical outcomes across patient cohorts . This comprehensive approach will not only identify the specific epigenetic mechanisms silencing PDLIM2 but also inform potential targeted epigenetic therapies to restore its tumor-suppressive functions.

How can PDLIM2 Antibody facilitate research into therapeutic combinations targeting both PDLIM2 restoration and immune checkpoint blockade?

PDLIM2 antibody is invaluable for developing and evaluating therapeutic strategies that combine PDLIM2 restoration with immune checkpoint blockade. In preclinical studies, researchers should first establish baseline PDLIM2 expression in tumor models using western blotting and immunohistochemistry with the HRP-conjugated antibody . Subsequently, implement a factorial design testing epigenetic drugs (DNA methyltransferase inhibitors, histone deacetylase inhibitors) alone or in combination with anti-PD-1/PD-L1 antibodies, monitoring PDLIM2 restoration via immunoblotting at multiple timepoints .

For mechanistic understanding, analyze how PDLIM2 restoration affects PD-L1 expression on tumor cells (which can be induced by chemotherapeutic and epigenetic drugs) using flow cytometry and immunohistochemistry . Additionally, perform ChIP-seq analysis to examine how PDLIM2 restoration alters genome-wide binding patterns of NF-κB/RelA and STAT3, focusing on genes involved in antigen presentation and T-cell activation .

In translational research, establish patient-derived xenograft models and test optimized drug combinations while monitoring PDLIM2 expression, immune infiltration, and tumor response. Use sequential tumor biopsies during treatment to correlate PDLIM2 restoration (detected by immunohistochemistry) with changes in the tumor immune microenvironment and clinical response . This systematic approach will establish the mechanistic basis for synergy between PDLIM2-restoring therapies and immune checkpoint blockade, potentially leading to more effective combination therapies for cancer treatment.