PDLIM7 Antibody

What is PDLIM7 and what structural features characterize this protein?

PDLIM7 (PDZ and LIM domain protein 7) is a 457 amino acid protein with a molecular mass of 49.8 kDa that functions primarily as a scaffold for coordinated protein assembly. The protein contains two key structural domains:

• A PDZ domain that facilitates protein-protein interactions

• LIM domains that enable binding to actin filaments in both skeletal muscle and non-muscle tissues

PDLIM7 localizes primarily to the cytoplasm and exists in up to six different isoforms. It is notably expressed in heart and skeletal muscle tissue and serves as a cellular marker for characterizing astrocytes. Common synonyms include LMP3, LMP, LMP1, and ENIGMA .

The protein plays essential roles in:

• Localizing LIM-binding proteins to actin filaments

• Maintaining proper cellular architecture

• Direct and endochondral bone formation

• Fracture repair processes

• BMP6 signaling pathway

What applications are PDLIM7 antibodies optimized for, and what are their technical specifications?

PDLIM7 antibodies are optimized for multiple experimental applications with varying technical specifications:

Most commercially available PDLIM7 antibodies are rabbit polyclonal antibodies in unconjugated format, with observed molecular weights ranging from 48-55 kDa .

For optimal results, antibodies should be stored at -20°C in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3). Under these conditions, they typically remain stable for one year after shipment .

How should researchers optimize antigen retrieval when performing immunohistochemistry with PDLIM7 antibodies?

Optimizing antigen retrieval is critical for successful PDLIM7 immunodetection in tissue samples. Based on validation data:

Primary Recommendation:

• Use TE buffer at pH 9.0 for optimal epitope exposure in FFPE tissue sections

Alternative Method:

• Citrate buffer at pH 6.0 can also be effective but may yield lower signal intensity compared to TE buffer

The optimization protocol should include:

• Deparaffinization of tissue sections completely using xylene

• Rehydration through graded alcohols to water

• Heat-induced epitope retrieval using TE buffer (pH 9.0) in a pressure cooker or microwave

• Cooling slides to room temperature gradually

• Blocking with appropriate blocking reagent to minimize background

• Primary antibody incubation at dilutions ranging from 1:20-1:800, depending on tissue type

• Visualization using appropriate detection systems

For difficult tissues with high background, additional optimization steps may include:

• Extended blocking times

• Reduced primary antibody concentration

• Inclusion of detergents in washing steps

• Titration experiments to determine optimal antibody concentration for each tissue type

How does PDLIM7 interact with PDLIM2 to regulate NF-κB signaling pathways?

PDLIM7 and PDLIM2 form heterodimeric E3 ligases that synergistically inhibit NF-κB signaling through a multi-step process:

• Heterodimer Formation: PDLIM7 directly binds to PDLIM2 through specific domains (demonstrated through co-immunoprecipitation experiments)

• Ubiquitination Mechanisms: PDLIM7 promotes K63-linked polyubiquitination of PDLIM2, enhancing its activity

• p65 Degradation Pathway:

  • PDLIM7 directly binds to p65 and mediates its polyubiquitination

  • The p62/Sqstm1 protein binds to both polyubiquitinated PDLIM2 (via UBA domain) and the proteasome (via PB1 domain)

  • This interaction facilitates transfer of the NF-κB-PDLIM2 complex to the proteasome

  • The result is enhanced p65 degradation

• Functional Consequences:

  • Knockdown of PDLIM7 increases nuclear p65 protein levels without affecting cytoplasmic p65 or IκBα degradation

  • Double knockdown of PDLIM7 and PDLIM2 results in greater accumulation of nuclear p65 than single knockdowns

  • Cells lacking both PDLIM7 and PDLIM2 show enhanced production of proinflammatory cytokines (IL-6, IL-12β, G-CSF)

The critical domains for this interaction include the LIM domains of PDLIM7, as demonstrated through deletion mutation experiments .

What role does PDLIM7 play in cancer progression, particularly in castration-resistant prostate cancer?

PDLIM7 has emerged as a significant factor in cancer progression, with particularly important roles in castration-resistant prostate cancer (CRPC):

• PDLIM7 Expression in CRPC:

  • PDLIM7 is upregulated in CRPC cells compared to hormone-sensitive prostate cancer

  • H3K27 acetylation activates PDLIM7 expression in these cells

  • CBP/p300 increases H3K27ac levels in the PDLIM7 promoter region

• Functional Effects in CRPC:

  • Knockdown of PDLIM7 suppresses CRPC cell proliferation, migration, and angiogenesis

  • PDLIM7 silencing enhances sensitivity of CRPC cells to docetaxel treatment

  • PDLIM7 modulates O-GlcNAc levels in CRPC cells

• Mechanistic Pathway:

  • PDLIM7 was identified through peptidome screening of CRPC patient serum

  • Among several candidates (including USP46, TUBB4B, and afamin), only PDLIM7 knockdown significantly altered CRPC cell viability

  • PDLIM7 affects downstream targets involved in cell proliferation and survival pathways

• Therapeutic Implications:

  • PDLIM7 represents a potential molecular target for treating CRPC

  • Combined targeting of PDLIM7 and conventional chemotherapy might improve treatment outcomes

  • PDLIM7 inhibition could potentially overcome docetaxel resistance in CRPC

The prognostic value of PDLIM7 has also been demonstrated in other cancers, including acute myeloid leukemia, hepatocellular carcinoma, and gastric cancer .

What are the optimal protocols for subcellular fractionation when studying PDLIM7 localization and function?

When studying PDLIM7's subcellular localization and nuclear-cytoplasmic distribution, proper fractionation is essential. The following protocol has been validated for PDLIM7 research:

Recommended Fractionation Protocol:

• Cell Preparation:

  • Harvest cells at 70-80% confluence

  • Wash twice with ice-cold PBS

  • Collect by gentle scraping or trypsinization

• Fractionation Steps:

  • Separate into three distinct fractions:

    • Cytoplasmic fraction

    • Nuclear soluble fraction

    • Nuclear insoluble fraction

• Fraction Validation:

  • Confirm purity using the following markers:

    • Cytoplasmic fraction: cdc37

    • Nuclear soluble fraction: LSD1

    • Nuclear insoluble fraction: Histone H3

• PDLIM7 Distribution Analysis:

  • PDLIM7 is typically detected in all three fractions

  • p65 degradation primarily occurs in the nuclear insoluble fraction

  • For knockdown studies, evaluate changes in all fractions to fully understand PDLIM7's role

This fractionation approach has been successfully used to demonstrate that PDLIM7 knockdown increases nuclear p65 levels without affecting cytoplasmic p65, suggesting PDLIM7's role in nuclear degradation rather than cytoplasmic retention or nuclear translocation .

How should researchers design controls when evaluating PDLIM7 knockdown effects in cellular systems?

Proper control design is critical for accurate interpretation of PDLIM7 knockdown experiments. Based on published methodologies:

Essential Controls for PDLIM7 Knockdown Experiments:

• siRNA Controls:

  • Non-targeting siRNA with similar GC content to PDLIM7 siRNA

  • Multiple independent PDLIM7-targeting siRNAs to rule out off-target effects

  • Validation of knockdown efficiency by both RT-qPCR and Western blot

• Functional Validation Controls:

  • For inflammatory response studies:

    • Time-course analysis (1h, 2.5h, 5h post-stimulation) to capture temporal dynamics

    • Multiple cytokine measurements (IL-6, IL-12β, TNFα, G-CSF, CXCL-2, CXCL-10)

    • LPS or other stimulant concentration curves

  • For cancer studies:

    • Cell viability assays with appropriate time points

    • Migration/invasion assays with proper matrix controls

    • Drug sensitivity tests with concentration gradients

• Combined Knockdown Controls:

  • Single knockdowns of PDLIM7, PDLIM2, or p62/Sqstm1 alongside double knockdowns

  • This approach reveals synergistic or redundant functions

  • Particularly important for studying heterodimeric relationships

• Rescue Experiments:

  • Expression of siRNA-resistant wild-type PDLIM7

  • Expression of domain deletion mutants (ΔLIM3, etc.)

  • These controls confirm specificity and identify functional domains

The search results demonstrate that cells lacking both PDLIM7 and either PDLIM2 or p62/Sqstm1 show enhanced production of proinflammatory cytokines compared to control cells or single knockdown cells, highlighting the importance of appropriate controls in multi-protein studies .

What approaches can researchers use to measure PDLIM7's E3 ligase activity in experimental settings?

Measuring PDLIM7's E3 ligase activity requires specialized approaches to assess ubiquitination dynamics:

Methodological Approaches for Assessing PDLIM7 E3 Ligase Activity:

• In Vivo Ubiquitination Assays:

  • Transfect cells with tagged ubiquitin constructs alongside PDLIM7

  • Immunoprecipitate target proteins (e.g., p65, PDLIM2)

  • Detect polyubiquitination by Western blot

  • Include proteasome inhibitors (MG132) to prevent degradation of ubiquitinated proteins

• Linkage-Specific Ubiquitination Analysis:

  • Use ubiquitin mutants (K48R, K63R) to determine ubiquitin chain types

  • PDLIM7 promotes K63-linked polyubiquitination of PDLIM2

  • This linkage type is critical for determining functional outcomes

• Domain Mapping for E3 Ligase Activity:

  • Generate deletion mutants of PDLIM7 (particularly the LIM domains)

  • Test each mutant's ability to mediate ubiquitination

  • Identify critical domains for E3 ligase activity

• Heterodimer E3 Ligase Activity:

  • Co-express PDLIM7 and PDLIM2 at varying ratios

  • Measure changes in target protein ubiquitination

  • Assess synergistic effects on ubiquitination and subsequent degradation

• Proteasomal Degradation Assessment:

  • Compare degradation rates in control vs. PDLIM7-overexpressing cells

  • Monitor nuclear vs. cytoplasmic degradation of target proteins

  • Use proteasome inhibitors as controls

These approaches collectively demonstrate that PDLIM7 functions both directly as an E3 ligase for p65 and as an enhancer of PDLIM2's E3 ligase activity through promoting its K63-linked polyubiquitination .

How can researchers troubleshoot non-specific binding when using PDLIM7 antibodies in various applications?

Non-specific binding is a common challenge when working with PDLIM7 antibodies. Here are evidence-based approaches to improve specificity:

Application-Specific Troubleshooting Strategies:

• Western Blot Optimization:

  • Increase blocking stringency (5% milk or commercial blockers)

  • Optimize primary antibody dilution (recommended range: 1:2000-1:10000)

  • Increase wash duration and frequency using TBST

  • Use immunoreaction enhancers like Can get Signal solution

  • Consider gradient gels to better separate proteins near PDLIM7's molecular weight (48-55 kDa)

• Immunohistochemistry Improvements:

  • Test different antigen retrieval methods (compare TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Titrate antibody concentration widely (1:20-1:800)

  • Include peptide competition controls to confirm specificity

  • Use PDLIM7 knockout/knockdown tissues as negative controls

  • Consider chromogenic vs. fluorescent detection systems

• Immunoprecipitation Refinements:

  • Pre-clear lysates with protein A/G beads

  • Use 0.5-4.0 μg antibody for 1.0-3.0 mg protein lysate

  • Include isotype control antibodies (e.g., rabbit IgG) for background assessment

  • Optimize wash buffer stringency

  • Consider crosslinking antibodies to beads to prevent IgG contamination

• Validation Using Multiple Methods:

  • Confirm results with at least two independent PDLIM7 antibodies

  • Validate findings using PDLIM7 knockdown/knockout systems

  • Use overexpression systems with tagged PDLIM7 as complementary approaches

Researchers have successfully used these approaches to demonstrate specific PDLIM7-PDLIM2 interactions through co-immunoprecipitation and to visualize nuclear vs. cytoplasmic PDLIM7 distribution .

What experimental design best differentiates between the various isoforms of PDLIM7?

Distinguishing between PDLIM7 isoforms requires specialized experimental approaches:

Isoform Differentiation Strategies:

• RT-PCR and qPCR:

  • Design primers spanning exon-exon junctions specific to each isoform

  • Use nested PCR for low-abundance isoforms

  • Perform quantitative analysis to determine relative expression levels

  • Include tissue-specific controls (heart and skeletal muscle express highest levels)

• Protein-Level Differentiation:

  • Use high-resolution gel systems (7.5-10% gradient gels)

  • Separate isoforms based on molecular weight differences

  • Consider 2D gel electrophoresis for isoforms with similar molecular weights but different isoelectric points

  • Use isoform-specific antibodies when available

• Mass Spectrometry:

  • Perform immunoprecipitation with PDLIM7 antibodies

  • Analyze by LC-MS/MS to identify unique peptides for each isoform

  • Quantify isoform-specific peptides for relative abundance measurement

  • This approach was successful in identifying PDLIM7 in peptidome analysis of CRPC patient serum

• Expression Constructs:

  • Generate tagged constructs of each PDLIM7 isoform

  • Express in cell lines with low endogenous PDLIM7

  • Compare functional outcomes (protein interactions, cellular localization)

  • Use for antibody validation and specificity testing

Up to six different isoforms have been reported for PDLIM7, with various tissue distribution patterns. Heart muscle and skeletal muscle show particularly high expression levels, making them suitable positive controls for isoform studies .

How do recent findings on PDLIM7's role in NF-κB signaling impact experimental design in inflammatory disease research?

Recent discoveries about PDLIM7's role in NF-κB signaling have significant implications for inflammatory disease research design:

Experimental Design Considerations Based on PDLIM7-NF-κB Insights:

• Cellular Compartment Analysis:

  • Design experiments to specifically examine nuclear vs. cytoplasmic effects

  • Include subcellular fractionation (cytoplasmic, nuclear soluble, nuclear insoluble)

  • Monitor both nuclear translocation and nuclear degradation of NF-κB components

  • PDLIM7 primarily affects nuclear degradation of p65 rather than its translocation

• Temporal Dynamics:

  • Implement time-course analyses (1h, 2.5h, 5h post-stimulation)

  • The enhancement of proinflammatory cytokine expression in PDLIM7 knockdown cells is most prominent at 5h post-stimulation

  • This suggests PDLIM7's regulatory effects are more critical for the resolution phase of inflammation

• Protein Complex Analysis:

  • Include co-immunoprecipitation studies to detect PDLIM7-PDLIM2 heterodimers

  • Analyze p62/Sqstm1 interactions with both polyubiquitinated PDLIM2 and the proteasome

  • Assess how these interactions change during inflammatory responses

  • Consider proximity ligation assays to visualize these interactions in situ

• Cytokine Expression Profiling:

  • Measure multiple NF-κB-dependent cytokines (IL-6, IL-12β, TNFα, G-CSF, CXCL-2, CXCL-10)

  • Include both early and late-phase inflammatory mediators

  • Analyze at both mRNA and protein levels

  • PDLIM7 knockdown enhances expression of multiple inflammatory cytokines

• Synergistic Effects:

  • Design studies to examine interactions between PDLIM7, PDLIM2, and p62/Sqstm1

  • Include single and combined knockdowns/knockouts

  • Analyze additive or synergistic effects on inflammatory responses

  • Double knockdown of PDLIM7 and PDLIM2 shows greater effects than single knockdowns

These considerations can significantly enhance the translational relevance of PDLIM7 research in inflammatory conditions, potentially leading to novel therapeutic approaches targeting this regulatory pathway.