PLEKHA7 Antibody

What is PLEKHA7 and why is it important in cell biology research?

PLEKHA7 is a pleckstrin homology domain-containing protein that plays a crucial role in zonula adherens (ZA) biogenesis and maintenance. In humans, the canonical PLEKHA7 protein consists of 1121 amino acid residues with a molecular mass of approximately 127.1 kDa . The protein contains two WW domains and one pleckstrin homology (PH) domain in its N-terminal half, while the C-terminal portion features coiled-coil and proline-rich domains .

PLEKHA7 is particularly important because it has a distinct localization at adherens junctions that differs from other junction proteins. Unlike most adherens junction markers that distribute along the lateral regions of polarized epithelial cells, PLEKHA7 is concentrated specifically in the apical junctional belt, similar to afadin . This specialized localization makes PLEKHA7 antibodies valuable tools for distinguishing different junctional complexes and studying junction-specific functions.

What applications are PLEKHA7 antibodies commonly used for?

PLEKHA7 antibodies are utilized across several experimental applications:

• Immunofluorescence (IF): The most widely used application, allowing visualization of PLEKHA7's distinct localization at the zonula adherens .

• Western Blotting (WB): Used to detect PLEKHA7 protein in cell and tissue lysates, typically identifying major polypeptides of approximately 135 kDa and 145 kDa .

• Immunohistochemistry (IHC): Used in both frozen (IHC-fr) and paraffin-embedded (IHC-p) tissue sections to examine PLEKHA7 distribution in different epithelial tissues .

• Immunocytochemistry (ICC): Applied to cultured cells to study PLEKHA7 localization and dynamics .

• Immunoelectron Microscopy: Provides ultrastructural localization of PLEKHA7 at adherens junctions, showing its precise positioning approximately 28 nm from the plasma membrane .

• Co-immunoprecipitation: Used to identify PLEKHA7 interacting partners, such as components of RNA-binding protein complexes and actin-related proteins .

In which tissues and cell types is PLEKHA7 expression most prominent?

PLEKHA7 is widely expressed across many epithelial tissues. Immunofluorescence studies have detected PLEKHA7 at epithelial junctions in multiple organs:

• Kidney (except glomeruli, where it is notably absent)

• Liver

• Pancreas

• Intestine (including colon)

• Retina

• Cornea

Northern blotting analysis has identified two distinct PLEKHA7 transcripts (approximately 5.5 kb and 6.5 kb) in epithelial tissues . In cultured cells, PLEKHA7 antibodies consistently label the junctional regions of kidney epithelial cell lines such as MDCK (canine) and mpkCCDc14 (mouse) .

How do researchers validate the specificity of PLEKHA7 antibodies?

Validation of PLEKHA7 antibodies typically involves multiple complementary approaches:

• Knockdown/Knockout Verification: Comparing antibody signal in control cells versus cells with PLEKHA7 depletion through shRNA or CRISPR/Cas9 methods. For example, researchers have demonstrated decreased signal of the ~145 kDa polypeptide in MDCK cells depleted of PLEKHA7 by shRNA-mediated interference .

• Antigen Mapping: Determining the specific epitope recognized by antibodies using bacterially expressed fragments of the PLEKHA7 protein .

• Multiple Antibody Concordance: Using different antibodies targeting distinct regions of PLEKHA7 to confirm consistent localization patterns.

• Expected Subcellular Localization: Verifying that the antibody detects PLEKHA7 at its known subcellular locations, particularly the apical junctional belt of epithelial cells .

How can PLEKHA7 antibodies help investigate its role in microRNA regulation?

Recent research has uncovered a fascinating role for PLEKHA7 in microRNA-mediated gene regulation at the zonula adherens. PLEKHA7 antibodies can be used to:

• Examine RISC Component Co-localization: Immunofluorescence with PLEKHA7 antibodies combined with AGO2 (Argonaute 2) staining reveals their co-localization at the zonula adherens, suggesting a role for PLEKHA7 in recruiting miRNA processing machinery to cell junctions .

• Analyze Protein Interactions: Co-immunoprecipitation experiments using PLEKHA7 antibodies have identified interactions with RNA-binding proteins and RISC components, revealing a significant enrichment for RNA-binding proteins among PLEKHA7 interactors .

• Study miRNA Loading: RNA immunoprecipitation studies show that PLEKHA7 depletion affects AGO2 loading of specific miRNAs (miR-24, miR-200c, and miR-203a), and similar results are observed upon knockdown of PLEKHA7-associated LIM domain proteins . PLEKHA7 antibodies can help track these changes in miRNA regulation.

• Investigate Junction-Specific Gene Regulation: By using PLEKHA7 antibodies to differentiate junctional populations of miRNA regulatory complexes from cytoplasmic ones, researchers can explore compartment-specific gene regulation mechanisms.

What role does PLEKHA7 play in the relationship between adherens junctions and the actin cytoskeleton?

PLEKHA7 antibodies have been instrumental in uncovering the critical connections between adherens junctions and actin organization:

• Actomyosin Organization Analysis: Gene ontology enrichment analysis of PLEKHA7-interacting proteins reveals significant enrichment for processes and functions related to the actin cytoskeleton . Immunofluorescence using PLEKHA7 antibodies combined with actin visualization techniques demonstrates that PLEKHA7 knockout cells exhibit disrupted actomyosin organization at the zonula adherens, with disorganized circumferential actin rings and diffused junctional Myosin IIB .

• Identification of Actin-Related Interactors: Proteomic analyses following PLEKHA7 immunoprecipitation have identified several LIM domain-containing proteins among PLEKHA7 interactors, including LMO7, LIMCH1, and PDLIM1, which are known to have actin-binding functions .

• Junction Stability Assessment: PLEKHA7 antibodies can be used to examine how perturbations to the actin cytoskeleton affect adherens junction integrity, helping researchers understand the bidirectional relationship between junctions and the cytoskeleton.

How can researchers use PLEKHA7 antibodies to differentiate between zonula adherens and other junctional complexes?

PLEKHA7 has a uniquely specific localization pattern that makes its antibodies valuable for distinguishing different junctional domains:

• Differential Junctional Labeling: Unlike most adherens junction proteins (E-cadherin, p120 catenin, β-catenin, and α-catenin) that distribute along the lateral regions of polarized epithelial cells, PLEKHA7 is concentrated specifically in the apical junctional belt . This makes PLEKHA7 antibodies excellent markers for specifically identifying the zonula adherens.

• Distinction from Tight Junctions: PLEKHA7's tissue distribution and subcellular localization are distinctly different from tight junction markers like ZO-1 . For example, PLEKHA7 is not detected within kidney glomeruli, whereas ZO-1 is present in this location.

• Triple Labeling Experiments: Combining PLEKHA7 antibodies with markers for tight junctions (ZO-1) and lateral adherens junctions (E-cadherin) in triple-labeling experiments allows precise mapping of different junction types in complex epithelial tissues.

• Super-Resolution Microscopy: Using PLEKHA7 antibodies in super-resolution microscopy applications can reveal the precise nanoscale organization of the zonula adherens relative to other junctional complexes.

What are the optimal conditions for PLEKHA7 detection in immunofluorescence applications?

For optimal immunofluorescence detection of PLEKHA7:

• Fixation Method: Paraformaldehyde fixation (typically 4%) preserves PLEKHA7 epitopes while maintaining cellular architecture. For detailed analysis of junction-cytoskeleton relationships, methanol fixation may better preserve cytoskeletal components while still allowing PLEKHA7 detection .

• Permeabilization: Gentle permeabilization with 0.1-0.2% Triton X-100 is typically sufficient for antibody access to junctional PLEKHA7.

• Blocking Solution: 5% normal serum (matched to the species of the secondary antibody) with 1% BSA helps reduce background staining.

• Antibody Concentration: Primary PLEKHA7 antibodies are typically used at 1:100 to 1:500 dilutions, though optimal concentration should be determined empirically for each antibody and application .

• Incubation Time: Overnight incubation at 4°C often yields the best signal-to-noise ratio for junctional proteins like PLEKHA7.

• Co-staining Considerations: When co-staining with other junctional markers, sequential rather than simultaneous incubation may be necessary if antibodies are raised in the same species.

What are common pitfalls when using PLEKHA7 antibodies and how can they be addressed?

Several challenges may arise when working with PLEKHA7 antibodies:

• Junction Disruption During Processing: Adherens junctions can be disrupted during sample preparation. Using calcium-containing buffers throughout the fixation and staining process helps maintain junction integrity and PLEKHA7 localization.

• Specificity Issues: Some PLEKHA7 antibodies may cross-react with related proteins. Validation using PLEKHA7 knockdown/knockout samples is essential for confirming specificity .

• Isoform Detection: PLEKHA7 has multiple isoforms (up to 3 different isoforms reported), and not all antibodies detect all isoforms equally . Western blot analysis typically detects major polypeptides of approximately 135 kDa and 145 kDa, corresponding to different isoforms .

• Cell Confluence Effects: PLEKHA7 localization is highly dependent on cell confluence and junction maturity. Performing experiments with cells at consistent confluence levels (typically fully confluent for 2-3 days) ensures reproducible results.

• Background Signal: Non-specific background can obscure junctional staining. Extended blocking steps (2+ hours) and thorough washing (at least 3 x 10 minutes) can significantly improve signal-to-noise ratio.

What is the optimal experimental design for co-immunoprecipitation using PLEKHA7 antibodies?

For successful co-immunoprecipitation experiments with PLEKHA7 antibodies:

• Lysis Conditions: Use mild lysis buffers (e.g., containing 0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions. Including phosphatase and protease inhibitors is crucial to prevent degradation of PLEKHA7 and its interacting partners.

• Antibody Selection: Choose PLEKHA7 antibodies validated for immunoprecipitation applications. Monoclonal antibodies targeting specific epitopes (e.g., within residues 920-1020 of human PLEKHA7) have been successfully used for immunoprecipitation studies .

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding before adding the PLEKHA7 antibody.

• Controls: Include an isotype control antibody immunoprecipitation to distinguish between specific and non-specific interactions.

• Cross-linking Consideration: For studying weaker or transient interactions, mild crosslinking before lysis (e.g., with DSP or formaldehyde) may help preserve complexes.

• Verification: Confirm successful immunoprecipitation by immunoblotting a portion of the precipitate for PLEKHA7 before analyzing co-precipitating proteins .

• Interaction Validation: Validate novel interactions through reciprocal co-immunoprecipitation and additional techniques like proximity ligation assay.

How can quantitative analysis of PLEKHA7 immunostaining provide insights into junction stability?

Quantitative analysis of PLEKHA7 immunofluorescence can reveal important information about junction dynamics:

What does altered PLEKHA7 localization indicate about the roles of LIM domain proteins in junction organization?

Analysis of PLEKHA7 localization in conjunction with LIM domain proteins has revealed important insights:

• Sequential Dependency: While knockdown of LIM domain proteins (LMO7, LIMCH1, or PDLIM1) results in loss of junctional AGO2 localization, it does not affect PLEKHA7 localization . Conversely, PLEKHA7 knockout does not affect the junctional localization of these LIM proteins . This suggests a pathway where PLEKHA7 and LIM proteins independently localize to junctions, but both are required for AGO2 recruitment.

• Distinct Actin Phenotypes: Different LIM protein knockdowns produce distinct effects on actomyosin organization at the zonula adherens, which can be observed in relation to PLEKHA7 localization :

  • LMO7 knockdown: Loose, multifurcated actin cables

  • LIMCH1 knockdown: Severe fragmentation or absence of the apical actin ring

  • PDLIM1 knockdown: Jagged actomyosin cables with intracellular aggregates

• Junction-Cytoskeleton Interface: The relationship between PLEKHA7 and these LIM proteins helps define the interface between adherens junctions and the actin cytoskeleton, with implications for understanding how junctional stability is maintained.

How can researchers interpret differences in PLEKHA7 expression and localization across different tissues?

Interpreting tissue-specific patterns of PLEKHA7 expression requires consideration of several factors:

• Tissue Architecture Correlation: PLEKHA7's distribution pattern often correlates with the specific architectural organization of different epithelia. For example, its absence from kidney glomeruli compared to its presence in tubular epithelium reflects fundamental differences in junction organization between these structures .

• Junction Type Differentiation: In tissues with complex junction arrangements, PLEKHA7 specifically marks the zonula adherens, distinguishing this structure from other adherens junctions along the lateral membrane .

• Developmental Context: Changes in PLEKHA7 expression or localization during development may indicate critical periods of junction remodeling and epithelial maturation.

• Pathological Alterations: Alterations in PLEKHA7 expression or localization in disease states may provide insights into pathological mechanisms involving junction disruption.

• Comparative Analysis: Comparing PLEKHA7 patterns with other junction proteins (e.g., ZO-1, E-cadherin) across tissues can reveal tissue-specific junction specializations and potential functional differences.

How might PLEKHA7 antibodies contribute to understanding the dynamics of junction assembly and disassembly?

PLEKHA7 antibodies offer promising applications for studying junction dynamics:

• Live-Cell Imaging: Combining PLEKHA7 antibody fragments or nanobodies with live-cell imaging techniques could allow real-time visualization of zonula adherens dynamics during junction formation and remodeling.

• Pulse-Chase Experiments: Using PLEKHA7 antibodies in pulse-chase experiments could help determine the turnover rate of PLEKHA7 at adherens junctions and how this changes under different conditions.

• Super-Resolution Microscopy: Applying PLEKHA7 antibodies in super-resolution microscopy techniques can reveal the nanoscale organization and dynamics of the zonula adherens with unprecedented detail.

• Correlative Light-Electron Microscopy: Combining PLEKHA7 immunofluorescence with electron microscopy could provide insights into the ultrastructural changes during junction assembly and disassembly.

• Optogenetic Approaches: Using PLEKHA7 antibodies to validate optogenetic tools that manipulate PLEKHA7 function could enable precise temporal control over junction dynamics for mechanistic studies.

What emerging roles of PLEKHA7 in RNA regulation could be investigated with PLEKHA7 antibodies?

The connection between PLEKHA7 and RNA regulation opens several exciting research avenues:

• Junction-Specific Transcriptome Analysis: PLEKHA7 antibodies could be used for proximity-based RNA labeling to identify transcripts specifically regulated at the zonula adherens.

• miRNA-Target Interaction Studies: Combining PLEKHA7 immunoprecipitation with RNA crosslinking techniques could help identify miRNA-target interactions specifically occurring at PLEKHA7-containing complexes.

• RNA Granule Association: Investigating the potential role of PLEKHA7 in organizing RNA granules at cell junctions through co-localization studies with RNA granule markers.

• Post-Transcriptional Regulation: Exploring how PLEKHA7-associated miRNA complexes regulate local translation at cell junctions through combined immunofluorescence and translation reporter assays.

• Signaling-Dependent RNA Regulation: Investigating how extracellular signals affect PLEKHA7-mediated RNA regulation through analysis of PLEKHA7 complex composition under different signaling conditions.

How can PLEKHA7 antibodies help elucidate its potential roles in epithelial pathologies?

PLEKHA7 antibodies could provide valuable insights into epithelial pathologies:

• Cancer Progression Analysis: Examining changes in PLEKHA7 expression and localization during epithelial-to-mesenchymal transition and cancer progression through tissue microarray analysis.

• Junction Integrity in Inflammatory Diseases: Investigating how inflammatory conditions affect PLEKHA7-dependent junction integrity in conditions like inflammatory bowel disease or asthma.

• Developmental Disorders: Studying potential roles of PLEKHA7 in developmental disorders involving epithelial morphogenesis through analysis of patient samples or developmental models.

• Biomarker Development: Exploring the potential of PLEKHA7 as a diagnostic or prognostic biomarker for conditions involving junction disruption through quantitative analysis of PLEKHA7 in patient samples.

• Therapeutic Target Validation: Using PLEKHA7 antibodies to validate the efficacy of potential therapeutic approaches targeting junction stability in epithelial pathologies.