PLEKHA7 Antibody, FITC conjugated

What is PLEKHA7 and what is its cellular function?

PLEKHA7 (Pleckstrin Homology Domain Containing, Family A Member 7) is a cytoplasmic protein that functions as a component of the epithelial adherens junction belt. It plays a critical role in linking the E-cadherin-p120ctn complex to the microtubule cytoskeleton . Structurally, PLEKHA7 contains two WW domains and one pleckstrin homology (PH) domain in its N-terminal half, along with coiled-coil and proline-rich domains in the C-terminal portion .

PLEKHA7 has been identified as having specific Rac1/Cdc42 GAP (GTPase-activating protein) activity, which modulates cell migration and blood-aqueous barrier function . Its expression is essential for zonula adherens biogenesis and maintenance, where it acts through interaction with KIAA1543/Nezha to anchor microtubules at their minus-ends to the zonula adherens .

What is the subcellular localization of PLEKHA7?

PLEKHA7 displays a highly specific subcellular localization at adherens junctions in epithelial tissues. Immunoelectron microscopy has definitively established that PLEKHA7 is localized at the adherens junctions in colonic epithelial cells, at a mean distance of approximately 28 nm from the plasma membrane .

Unlike other adherens junction markers such as E-cadherin, p120ctn, β-catenin, and α-catenin which are found along the lateral region of polarized epithelial cells, PLEKHA7 is concentrated specifically in the apical junctional belt, similar to afadin . This distinctive localization pattern suggests a specialized role in junctional complex organization.

What is the molecular weight of PLEKHA7 protein?

Immunoblotting analysis of PLEKHA7 consistently reveals major polypeptides with apparent molecular weights of approximately 135 kDa and 145 kDa in lysates of various cells and tissues . In some tissues such as pancreas, lung, eye, and liver, a larger polypeptide of approximately 240 kDa has also been detected, although this may represent cross-reactivity with an unrelated protein .

The detection of multiple transcripts (approximately 5.5 kb and 6.5 kb) by Northern blot analysis further supports the existence of multiple isoforms of PLEKHA7 .

What applications are suitable for PLEKHA7 antibody, FITC conjugated?

The FITC-conjugated PLEKHA7 antibody is suitable for several research applications:

• Western Blotting (WB)

• Immunofluorescence on cultured cells (IF (cc))

• Immunofluorescence on paraffin-embedded sections (IF (p))

• ELISA

The antibody has been validated for detecting human PLEKHA7, with specific targeting of amino acids 1001-1121 in the PLEKHA7 protein sequence .

How does PLEKHA7 modulate epithelial tight junction barrier function?

PLEKHA7 plays a crucial role in modulating epithelial tight junction (TJ) barrier function through several mechanisms:

• E-cadherin complex stabilization: Expression of PLEKHA7 constructs enhances recruitment of E-cadherin and associated proteins at the apical zonula adherens (ZA) and lateral puncta adherentia (PA) .

• Barrier dynamics regulation: PLEKHA7 affects the dynamics of assembly and disassembly of the TJ barrier. Studies with inducible PLEKHA7 expression showed decreased transepithelial resistance (TER) at 18 hours after assembly at normal calcium, and an attenuation in the fall in TER after extracellular calcium removal .

• Microtubule-dependent mechanisms: The attenuation in TER decrease after calcium removal is inhibited when cells are treated with nocodazole, indicating microtubule involvement in PLEKHA7's function .

• Protein complex formation: PLEKHA7 forms a complex with cytoplasmic TJ proteins ZO-1 and cingulin, and this association does not depend on the integrity of microtubules .

These findings suggest that PLEKHA7 modulates TJ barrier function through both E-cadherin protein complex-dependent and microtubule-dependent mechanisms.

What is the relationship between PLEKHA7 and primary angle closure glaucoma (PACG)?

PLEKHA7 has been identified as a susceptibility gene for primary angle closure glaucoma (PACG). Research has revealed several important aspects of this relationship:

• Downregulation in PACG: PLEKHA7 is downregulated in lens epithelial cells and iris tissue of PACG patients .

• Genetic association: PLEKHA7 expression correlates with the C risk allele of the sentinel SNP rs11024102, with risk allele carriers showing significantly reduced PLEKHA7 levels compared to non-risk allele carriers .

• Cytoskeletal effects: Silencing of PLEKHA7 in human immortalized non-pigmented ciliary epithelium (h-iNPCE) and primary trabecular meshwork cells affects actin cytoskeleton organization .

• Small GTPase regulation: PLEKHA7 specifically interacts with GTP-bound Rac1 and Cdc42 (but not RhoA), functioning as a novel Rac1/Cdc42 GAP that stimulates GTP hydrolysis without affecting nucleotide exchange .

• Barrier function: Silencing of PLEKHA7 compromises the paracellular barrier between h-iNPCE cells, consistent with the regulatory role of Rac1 and Cdc42 in maintaining tight junction permeability .

These findings suggest that downregulation of PLEKHA7 in PACG may affect blood-aqueous barrier integrity and aqueous humor outflow via its Rac1/Cdc42 GAP activity, contributing to disease etiology.

What controls should be included when using PLEKHA7 antibodies in immunofluorescence experiments?

When conducting immunofluorescence experiments with FITC-conjugated PLEKHA7 antibodies, the following controls should be included:

• Negative controls:

  • Isotype control (rabbit IgG) to assess non-specific binding

  • Secondary antibody only control (when using unconjugated primary antibodies)

  • PLEKHA7-depleted cells (using shRNA-mediated interference)

• Positive controls:

  • Known PLEKHA7-expressing epithelial cell lines like MDCK or mpkCCDc14 cells

  • Tissues with confirmed PLEKHA7 expression such as kidney cortex (tubules), pancreatic acini and ductules, liver (bile canaliculi), and intestinal epithelia

• Co-localization markers:

  • E-cadherin, p120ctn, β-catenin, and α-catenin (other adherens junction markers)

  • Differential markers: ZO-1 (which has distinct localization from PLEKHA7 in some tissues)

• Technical validation:

  • Peptide competition experiments using the immunizing antigen (KLH conjugated synthetic peptide derived from human PLEKHA7)

  • Varying antibody dilutions to determine optimal signal-to-noise ratio

What are the optimal protocols for using FITC-conjugated PLEKHA7 antibodies in immunofluorescence studies?

For optimal results when using FITC-conjugated PLEKHA7 antibodies for immunofluorescence:

For cultured cells:

• Fix cells with 4% paraformaldehyde in PBS for 15-20 minutes at room temperature

• Permeabilize with 0.1-0.2% Triton X-100 in PBS for 5-10 minutes

• Block with 1-3% BSA in PBS for 30-60 minutes

• Apply FITC-conjugated PLEKHA7 antibody at recommended dilution (typically 1:50 to 1:200) in blocking buffer

• Incubate overnight at 4°C in a humidified chamber

• Wash 3x with PBS

• Counterstain nucleus with DAPI

• Mount in anti-fade mounting medium

• Image using appropriate fluorescence microscope with FITC filter set (excitation ~495nm, emission ~519nm)

For tissue sections:

• For paraffin-embedded sections, perform antigen retrieval (typically citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Block endogenous fluorescence with 0.1-0.3% sodium borohydride

• Block with 5-10% normal serum in PBS with 0.1-0.3% Triton X-100

• Apply FITC-conjugated PLEKHA7 antibody at recommended dilution

• Incubate overnight at 4°C

• Wash extensively with PBS

• Counterstain and mount as above

How can researchers validate PLEKHA7 knockdown or overexpression models?

Validation of PLEKHA7 genetic manipulation requires multiple complementary approaches:

For knockdown validation:

• mRNA level assessment: RT-qPCR using PLEKHA7-specific primers

• Protein level assessment: Western blotting using anti-PLEKHA7 antibodies, looking for reduction in the 135-145 kDa bands

• Functional validation: Assess changes in:

  • Actin cytoskeleton organization (which is affected by PLEKHA7 silencing)

  • Transepithelial resistance measurements (as PLEKHA7 affects barrier function)

  • Paracellular permeability assays

• Phenotypic rescue: Re-introduction of shRNA-resistant PLEKHA7 to verify specificity

For overexpression validation:

• Protein detection: Western blotting showing increased intensity of PLEKHA7 bands

• Subcellular localization: Immunofluorescence confirming proper junctional localization of overexpressed protein

• Functional validation: Enhanced recruitment of E-cadherin and associated proteins at adherens junctions, altered TER dynamics, and modified response to calcium manipulation

How can PLEKHA7 antibodies be used to investigate adherens junction dynamics?

PLEKHA7 antibodies provide valuable tools for investigating adherens junction dynamics:

• Time-course experiments: Monitor PLEKHA7 recruitment during junction formation using calcium switch experiments, where FITC-conjugated antibodies allow for live-cell imaging

• Co-immunoprecipitation studies: Investigate protein complexes involving PLEKHA7, E-cadherin, p120ctn, and microtubule components

• Cytoskeletal disruption experiments: Assess the effects of nocodazole (microtubule disruptor) on PLEKHA7 localization and function

• GTPase activity assays: Examine the relationship between PLEKHA7 and Rac1/Cdc42 activity using PLEKHA7 antibodies in combination with GTPase activation assays

• FRAP (Fluorescence Recovery After Photobleaching): Using FITC-conjugated PLEKHA7 antibodies in live cells to measure the dynamic exchange of PLEKHA7 at junctions

What factors might affect PLEKHA7 antibody specificity and how can they be addressed?

Several factors can influence PLEKHA7 antibody specificity:

• Cross-reactivity: Some tissues may show labeling of larger polypeptides (~240 kDa) that might represent cross-reactivity with unrelated proteins . This can be addressed by:

  • Using multiple antibodies targeting different epitopes of PLEKHA7

  • Performing peptide competition assays

  • Validating with PLEKHA7 knockout/knockdown controls

• Isoform recognition: Multiple PLEKHA7 transcripts and protein isoforms exist (5.5 kb and 6.5 kb transcripts, 135 kDa and 145 kDa proteins) . Ensure the antibody recognizes the specific isoform of interest by:

  • Checking the specific epitope recognized by the antibody (e.g., AA 1001-1121)

  • Using positive control lysates with known isoform expression

• Fixation sensitivity: Different fixation methods may affect epitope accessibility. Optimize by:

  • Testing multiple fixation protocols (paraformaldehyde, methanol, acetone)

  • Performing antigen retrieval when necessary

  • Adjusting permeabilization conditions

How can researchers interpret changes in PLEKHA7 localization in disease models?

When interpreting alterations in PLEKHA7 localization in disease models:

• Distinguish redistribution from expression changes:

  • Compare immunofluorescence with total protein levels by Western blotting

  • Quantify fluorescence intensity at junctions versus cytoplasmic regions

• Correlate with junctional integrity:

  • Co-stain with other junction markers (E-cadherin, ZO-1)

  • Assess barrier function (TER measurements, permeability assays)

• Evaluate disease relevance:

  • In PACG models, correlate PLEKHA7 downregulation with changes in barrier function and actin cytoskeleton

  • Link observations to genetic variations (e.g., correlation with risk alleles)

• Consider tissue-specific patterns:

  • PLEKHA7 has tissue-specific distribution patterns distinct from other junction proteins

  • Some tissues (e.g., kidney glomeruli) naturally lack PLEKHA7 expression

Understanding these context-dependent variations is crucial for proper interpretation of experimental results.

How might PLEKHA7's Rac1/Cdc42 GAP activity be targeted in therapeutic approaches?

PLEKHA7's newly identified role as a Rac1/Cdc42 GAP offers potential therapeutic avenues:

• Small molecule modulators: Development of compounds that could enhance PLEKHA7's GAP activity might help restore proper barrier function in conditions like PACG

• Peptide-based approaches: Designing peptides that mimic PLEKHA7's interaction domains with Rac1/Cdc42 to modulate GTPase activity

• Gene therapy strategies: Restoring PLEKHA7 expression in tissues where it is downregulated, such as in PACG patients

• Combined cytoskeletal approaches: Targeting both microtubule and actin cytoskeleton regulation, given PLEKHA7's dual role in connecting adherens junctions to microtubules and affecting actin organization via Rac1/Cdc42

What technical advances might improve PLEKHA7 detection and functional analysis?

Future technical developments that could enhance PLEKHA7 research include:

• Super-resolution microscopy: Techniques like STORM or PALM could better resolve PLEKHA7's precise localization at the 28 nm distance from the plasma membrane

• Proximity labeling approaches: BioID or APEX2 fusions with PLEKHA7 to identify novel interaction partners at adherens junctions

• Optogenetic control: Light-inducible PLEKHA7 variants to study the temporal dynamics of junction assembly

• Improved antibody conjugates: Development of photoconvertible or multiple-wavelength antibody conjugates for longitudinal studies

• Nanobody development: Creation of PLEKHA7-specific nanobodies for improved tissue penetration and reduced background