PDLIM4 Antibody, FITC conjugated

What is PDLIM4 and what cellular functions does it serve?

PDLIM4 (PDZ and LIM Domain 4), also known as RIL, is an actin-associated protein that plays significant roles in cytoskeletal organization. The protein contains a PDZ domain and a LIM domain, which mediate protein-protein interactions. PDLIM4 has been shown to stimulate actin bundling by interacting with the actin-cross-linking protein α-actinin-1 and increasing its affinity to filamentous actin (F-actin) . This interaction promotes the formation of actin stress fibers, which are essential for cellular structure and motility. Additionally, PDLIM4 expression has been found to be reduced in several cancer cell lines, suggesting a potential tumor suppressor function .

In experimental approaches, researchers typically examine PDLIM4's subcellular localization, its binding partners (particularly α-actinin and F-actin), and phenotypic changes associated with its expression or silencing. Methodologically, this requires techniques such as immunofluorescence microscopy, co-immunoprecipitation, and functional assays measuring cell growth, migration, and cytoskeletal organization.

What distinguishes a FITC-conjugated PDLIM4 antibody from other labeled antibodies, and when is it most appropriate to use?

A FITC (Fluorescein Isothiocyanate)-conjugated PDLIM4 antibody has the fluorescent dye FITC directly attached to it, emitting green fluorescence when excited with appropriate wavelengths. The specific PDLIM4 antibody (AA 74-103) conjugated to FITC is generated from rabbits immunized with a KLH-conjugated synthetic peptide corresponding to amino acids 74-103 from the central region of human PDLIM4 .

This FITC-conjugated antibody is most appropriate for:

• Direct immunofluorescence microscopy: Eliminating the need for secondary antibodies, reducing background and cross-reactivity.

• Flow cytometry: For analyzing PDLIM4 expression in cell populations.

• Multiplexed imaging: When combined with antibodies conjugated to spectrally distinct fluorophores.

Methodologically, FITC-conjugated antibodies work optimally at pH >7.0 and should be protected from prolonged light exposure. When designing experiments, researchers should consider potential photobleaching and the relatively lower photostability of FITC compared to newer fluorophores like Alexa Fluor dyes. For optimal results, fixation protocols should be tested to ensure epitope preservation while maintaining cellular structure, particularly for cytoskeletal proteins like PDLIM4.

How should I optimize immunofluorescence protocols for studying PDLIM4's co-localization with actin structures?

Optimizing immunofluorescence protocols for PDLIM4 and actin co-localization requires careful consideration of fixation, permeabilization, and staining procedures:

Recommended protocol:

• Fixation: Use 4% paraformaldehyde for 15 minutes at room temperature to preserve cytoskeletal structures. Avoid methanol fixation, which can disrupt actin filaments.

• Permeabilization: Use 0.1% Triton X-100 for 5-10 minutes. For more delicate actin structures, consider 0.05% saponin.

• Blocking: Block with 5% BSA or 10% normal serum from a species different from the primary antibody source for 1 hour.

• Primary antibody incubation: Apply FITC-conjugated PDLIM4 antibody at 0.25-2 μg/mL concentration . Incubate overnight at 4°C.

• F-actin visualization: Since PDLIM4 has been shown to directly interact with F-actin , use rhodamine-phalloidin or Alexa Fluor 594-phalloidin to counterstain actin (spectrally distinct from FITC).

• Nuclear counterstaining: Use DAPI (blue) to avoid spectral overlap with FITC (green) and rhodamine/Alexa594 (red).

• Mounting: Use anti-fade mounting medium to minimize photobleaching of FITC.

For quantitative co-localization analysis, acquire Z-stack images and perform Pearson's correlation coefficient or Manders' overlap coefficient analysis. This approach is particularly valuable when studying how PDLIM4 regulates actin stress fiber formation and organization, as demonstrated in studies where PDLIM4 expression affects the morphology of actin cables .

What are the critical validation steps needed when using PDLIM4 antibodies in Western blotting applications?

When using PDLIM4 antibodies, including FITC-conjugated versions, for Western blotting, the following validation steps are essential:

• Positive and negative controls:

  • Positive control: Cell lines known to express PDLIM4 (non-cancerous epithelial cells)

  • Negative control: Cell lines with low PDLIM4 expression (certain prostate cancer cell lines like LNCaP, DU145, PC3)

  • PDLIM4 knockdown or knockout samples

• Detection of expected molecular weight: Full-length PDLIM4/RIL should appear at approximately 36 kDa. Be aware of the alternatively spliced isoform (RILaltCterm), which is 84 amino acids shorter .

• Antibody concentration optimization: For Western blotting, start with 0.04-0.4 μg/mL as recommended and adjust as needed.

• Loading control validation: Use housekeeping proteins (β-actin, GAPDH) while being mindful that PDLIM4 interacts with the actin cytoskeleton.

• Specificity testing:

  • Peptide competition assay using the immunizing peptide (amino acids 74-103)

  • Detection of overexpressed tagged PDLIM4 with both tag-specific and PDLIM4-specific antibodies

• Alternative antibody comparison: Compare results with another PDLIM4 antibody targeting a different epitope.

For FITC-conjugated antibodies in Western blotting, be aware that direct detection might have lower sensitivity than chemiluminescence. Consider using a secondary anti-FITC antibody conjugated to HRP to amplify the signal if needed. Document the exposure conditions carefully, as FITC can photobleach during imaging.

How can I effectively distinguish between the full-length PDLIM4 and its alternatively spliced isoform (RILaltCterm) in experimental systems?

Distinguishing between full-length PDLIM4/RIL and its alternatively spliced isoform RILaltCterm requires strategic experimental approaches due to several challenges:

• Antibody selection: The FITC-conjugated antibody targeting amino acids 74-103 will detect the full-length protein but may not recognize RILaltCterm if the epitope spans the alternatively spliced region. For comprehensive detection:

  • Use antibodies targeting the N-terminal PDZ domain (present in both isoforms)

  • Use C-terminal antibodies specific to either the LIM domain (full-length only) or the alternative C-terminus (RILaltCterm only)

• Western blotting optimization:

  • Use gradient gels (4-20%) to improve separation of the isoforms

  • Extended run times may be needed to resolve the 84-amino acid size difference

  • Be aware that RILaltCterm is extremely unstable due to its disordered C-terminal structure targeting it for rapid proteasomal degradation

• RT-PCR and qPCR approaches:

  • Design primers spanning exon junctions:

    • Primers spanning exons 5-6 junction: detect full-length only

    • Primers spanning exons 5-7 junction (skipping exon 6): detect RILaltCterm only

  • Perform quantitative PCR with isoform-specific primers for expression analysis

• Proteasome inhibition: When studying RILaltCterm, treat cells with proteasome inhibitors (MG132) to prevent its rapid degradation, as research has shown this isoform has a half-life of less than 1 hour due to ubiquitin-independent degradation in 20S proteasomes .

• Functional validation: The isoforms show distinct subcellular localization and effects on actin cytoskeleton:

  • Full-length PDLIM4: Forms thick fibrillar structures and promotes actin cable formation

  • RILaltCterm: Shows diffuse cytoplasmic localization with thin fibers in a mesh-like pattern

This comprehensive approach allows researchers to accurately distinguish and study both isoforms, which is critical given their opposing functions in actin cytoskeleton regulation.

What experimental approaches should be used to investigate PDLIM4's potential tumor suppressor function in cancer research?

Investigating PDLIM4's potential tumor suppressor function requires a multi-faceted experimental approach:

• Expression analysis in clinical samples:

  • Immunohistochemistry using PDLIM4 antibodies on tissue microarrays comparing normal vs. tumor tissues

  • Quantitative RT-PCR to measure PDLIM4 mRNA levels

  • Analysis of public databases (TCGA, GEO) for PDLIM4 expression correlation with patient survival

• Epigenetic regulation assessment:

  • Methylation-specific PCR of PDLIM4 promoter region

  • Treatment of cancer cell lines with DNA methyltransferase inhibitors (5-aza-2'-deoxycytidine) to assess PDLIM4 re-expression

• Functional studies in cell lines:

  • Re-expression experiments: Transfect PDLIM4 into cancer cell lines with low endogenous expression (like LNCaP, LAPC4, DU145, CWR22, and PC3 prostate cancer cells)

  • Phenotypic assays:

    • Proliferation (MTT/XTT, BrdU incorporation)

    • Clonogenicity (colony formation assay)

    • Cell cycle analysis (flow cytometry) to confirm G1 arrest

    • Apoptosis (Annexin V staining, TUNEL assay)

    • Migration and invasion (transwell assays)

    • Cytoskeletal organization (immunofluorescence)

• Mechanistic studies:

  • Immunoprecipitation with PDLIM4 antibodies to identify novel binding partners

  • F-actin binding assays to confirm direct interaction

  • Signaling pathway analysis (Western blotting for key signaling molecules)

• In vivo studies:

  • Xenograft models comparing tumor growth of control vs. PDLIM4-expressing cancer cells

  • Detailed analysis of tumor characteristics (proliferation, apoptosis, vascularization)

Data table: Expression and function of PDLIM4 in cancer cell lines

This integrated approach provides comprehensive evidence for PDLIM4's tumor suppressor role while elucidating the underlying mechanisms.

What are common challenges when using FITC-conjugated PDLIM4 antibodies in flow cytometry, and how can they be addressed?

When using FITC-conjugated PDLIM4 antibodies in flow cytometry, researchers encounter several challenges that require specific troubleshooting approaches:

• High background/non-specific binding:

  • Cause: Insufficient blocking or non-specific FITC binding

  • Solution: Increase blocking time (30-60 minutes) with 2-5% BSA or normal serum; include 0.1% Tween-20 in wash buffers; perform Fc blocking in primary cells

• Weak signal detection:

  • Cause: PDLIM4 is predominantly cytoplasmic and requires permeabilization; FITC has moderate brightness

  • Solution: Optimize permeabilization (0.1% saponin or 0.1% Triton X-100); adjust antibody concentration (use up to 2 μg/mL) ; consider longer incubation (overnight at 4°C)

• Photobleaching during acquisition:

  • Cause: FITC is prone to photobleaching

  • Solution: Minimize light exposure before acquisition; add anti-fade compounds to buffers; run samples immediately after preparation; adjust flow cytometer settings to minimize exposure time

• Spectral overlap:

  • Cause: FITC emission spectrum overlaps with other fluorophores

  • Solution: Perform proper compensation using single-stained controls; consider spectral unmixing if available; design panels to minimize spectral overlap with FITC

• Difficulty detecting PDLIM4's alternatively spliced isoform:

  • Cause: The RILaltCterm isoform is rapidly degraded

  • Solution: Pre-treat cells with proteasome inhibitors (MG132, 10μM for 4-6 hours); optimize fixation and permeabilization protocols to preserve protein integrity

• Variability in PDLIM4 expression based on cell state:

  • Cause: PDLIM4 expression and localization may change with cell cycle, stress conditions, or differentiation state

  • Solution: Synchronize cells when possible; use co-staining for cell cycle markers; document culture conditions precisely; include cytoskeletal markers for correlation analysis

For optimal results, researchers should validate their FITC-conjugated PDLIM4 antibody using Western blotting and immunofluorescence microscopy before flow cytometry applications. When presenting flow cytometry data, always include appropriate positive and negative controls, and document gating strategies thoroughly to support reproducibility of PDLIM4 expression analysis.

How should researchers interpret contradictory results when studying PDLIM4's interaction with α-actinin in different cell types?

When encountering contradictory results regarding PDLIM4's interaction with α-actinin across different cell types, researchers should consider the following interpretative framework and methodological approaches:

• Context-dependent interactions:

  • Research shows that while PDLIM4/RIL interacts with α-actinin-1 in some contexts , studies in breast cancer cells found that PDLIM4/RIL did not co-immunoprecipitate with α-actinin, unlike the related protein CLP36/PDLIM1

  • This suggests cell-type specific regulation of these interactions

• Methodological considerations for resolving contradictions:

  • Antibody epitope accessibility: Different antibodies may recognize distinct epitopes that could be masked in protein complexes

  • Co-IP conditions: Varying buffer strengths, detergents, and salt concentrations can preserve or disrupt protein interactions

  • Detection sensitivity: As demonstrated with CLP36-α-actinin interactions, different detection methods showed varying results (standard vs. high-sensitivity chemiluminescence)

• Experimental validation approach:

  • Reciprocal co-immunoprecipitation: Perform IP with α-actinin antibodies and blot for PDLIM4, and vice versa

  • Crosslinking: Use protein crosslinking before lysis to stabilize transient interactions

  • Proximity ligation assay (PLA): Detect protein interactions in situ with higher sensitivity than conventional co-localization

  • FRET or BiFC analysis: Directly measure protein proximity in living cells

  • Domain mapping: Use truncation mutants to identify which PDLIM4 domains mediate α-actinin binding

• Multiple isoform consideration:

  • The alternatively spliced isoform of PDLIM4 (RILaltCterm) lacks the LIM domain , which might be critical for α-actinin interaction

  • α-actinin itself exists in multiple isoforms (α-actinin-1 through 4) with tissue-specific expression patterns

• Data interpretation framework:

By applying this systematic approach to contradictory findings, researchers can better understand the nuanced, context-dependent nature of PDLIM4's interactions with α-actinin and its role in cytoskeletal regulation across different cellular environments.

What innovative experimental approaches could reveal PDLIM4's role in stress response pathways, particularly in relation to RILaltCterm stabilization during oxidative stress?

The discovery that RILaltCterm (the alternatively spliced isoform of PDLIM4) accumulates during oxidative stress opens exciting research directions requiring innovative methodologies:

• Real-time visualization of RILaltCterm stabilization:

  • Develop a split fluorescent protein system where one half is fused to RILaltCterm and the other to NQO1 (NAD(P)H quinone oxidoreductase 1), which blocks RILaltCterm degradation during oxidative stress

  • Create RILaltCterm reporter constructs with destabilized fluorescent proteins to monitor real-time accumulation during stress

  • Apply FRET-based biosensors to detect conformational changes in RILaltCterm under stress conditions

• Single-cell analysis of stress response dynamics:

  • Implement microfluidic platforms for controlled oxidative stress exposure with simultaneous imaging

  • Apply RNA-seq at single-cell resolution to correlate alternative splicing events with stress response

  • Use mass cytometry (CyTOF) with PDLIM4 isoform-specific antibodies to quantify cellular heterogeneity in stress response

• Proteomic profiling during stress conditions:

  • Apply SILAC or TMT labeling followed by mass spectrometry to identify stress-induced changes in the PDLIM4 interactome

  • Use BioID or APEX proximity labeling fused to RILaltCterm to identify stress-specific interaction partners

  • Perform global ubiquitinome analysis to understand how RILaltCterm evades ubiquitin-dependent degradation while undergoing ubiquitin-independent 20S proteasomal degradation

• Mechanistic studies of cytoskeletal remodeling under stress:

  • Apply live-cell super-resolution microscopy (SIM, STORM) to visualize RILaltCterm-mediated changes in actin dynamics during stress

  • Use optogenetic tools to induce local accumulation of RILaltCterm and observe immediate effects on cytoskeletal organization

  • Implement traction force microscopy to measure mechanical consequences of RILaltCterm-mediated cytoskeletal changes

• Translational approaches:

  • Develop cell-penetrating peptides mimicking the RILaltCterm C-terminal region to modulate actin cytoskeleton in disease contexts

  • Create small molecules targeting the RILaltCterm-NQO1 interaction to regulate stress response

  • Apply CRISPR-based strategies to modify exon 6 splicing to alter RILaltCterm/full-length PDLIM4 ratios

Proposed experimental workflow for studying RILaltCterm in stress response:

This comprehensive research strategy would significantly advance our understanding of how alternative splicing of PDLIM4 contributes to cellular adaptation during stress conditions.

How might researchers leverage PDLIM4 antibodies to explore the protein's differential roles in normal versus cancer cells, particularly in relation to actin cytoskeleton organization?

Researchers can strategically use PDLIM4 antibodies, including FITC-conjugated versions, to investigate its differential roles in normal versus cancer cells through the following integrated approaches:

• Comparative spatial proteomics:

  • Apply multiplexed immunofluorescence with FITC-conjugated PDLIM4 antibodies combined with actin markers and cancer-specific proteins

  • Implement tissue microarray analysis comparing normal tissues with matched tumors across cancer progression stages

  • Use CODEX or imaging mass cytometry for highly multiplexed protein detection to place PDLIM4 in its cytoskeletal and signaling context

• Differential interactome mapping:

  • Perform immunoprecipitation with PDLIM4 antibodies in normal versus cancer cells followed by mass spectrometry

  • Create BioID fusion constructs to identify proximity partners specific to each cellular context

  • Apply computational network analysis to identify cancer-specific PDLIM4 interaction subnetworks

• Cytoskeletal dynamics visualization:

  • Combine PDLIM4 immunostaining with live-cell actin probes to study dynamic differences

  • Implement correlative light and electron microscopy (CLEM) to examine ultrastructural details of PDLIM4-associated actin structures

  • Use FRAP (Fluorescence Recovery After Photobleaching) to measure PDLIM4 turnover rates at actin structures in normal vs. cancer cells

• Mechanical phenotyping:

  • Apply atomic force microscopy to measure cell stiffness changes associated with PDLIM4 expression

  • Use traction force microscopy to quantify cellular force generation

  • Implement microfluidic devices to assess cell deformability in relation to PDLIM4 status

• Functional rescue experiments:

  • Re-express PDLIM4 in cancer cells where it's downregulated and assess actin organization using immunofluorescence

  • Create domain-specific mutants to determine which regions are essential for cytoskeletal regulation

  • Apply optogenetic approaches to spatiotemporally control PDLIM4 activity and observe immediate effects on actin dynamics

Proposed experimental design for comparative analysis:

By systematically comparing these parameters between normal and cancer cells, researchers can elucidate how alterations in PDLIM4 contribute to cancer-associated cytoskeletal changes and identify potential therapeutic opportunities targeting these mechanisms. The combination of FITC-conjugated PDLIM4 antibodies with cutting-edge imaging and proteomic approaches offers unprecedented insights into context-dependent functions of this important cytoskeletal regulator.