klhl3 Antibody

What is KLHL3 and what are its key structural domains?

KLHL3 is a substrate adaptor protein that functions as part of a Cullin-RING E3 ubiquitin ligase (CRL) complex. The full-length protein contains three main structural domains: an N-terminal BTB domain that interacts with Cullin 3 (CUL3), a central BACK domain, and six C-terminal kelch-like repeats that are responsible for substrate recognition and binding. The kelch domain forms a β-propeller structure that serves as the substrate-binding interface, while the BTB domain mediates interaction with CUL3 to form a functional E3 ligase complex .

Which physiological pathways involve KLHL3?

KLHL3 primarily regulates blood pressure through modulation of the WNK-SPAK/OSR1 signaling pathway in the kidney. The CUL3-KLHL3 complex promotes ubiquitylation and degradation of WNK kinases, which in turn regulates the activity of ion co-transporters such as NCC (Na⁺/Cl⁻ co-transporter) and NKCC1 (Na⁺/K⁺/2Cl⁻ co-transporter 1) in the kidney's distal nephron . Recent studies have also revealed roles for KLHL3 in regulating paracellular chloride transport through interactions with claudin proteins , as well as potential involvement in energy metabolism, with KLHL3 deficiency showing protective effects against obesity, insulin resistance, and nonalcoholic fatty liver disease .

How is KLHL3 tissue distribution characterized?

KLHL3 exhibits varying expression levels across different tissues. Immunoblot and LacZ staining studies using KLHL3 knockout/LacZ knock-in mice have demonstrated that KLHL3 is strongly expressed in the brain and kidney, with weaker expression in the eye, testis, lung, heart, liver, stomach, and colon, while being virtually absent in muscle tissue . Within the kidney, KLHL3 is reportedly highly expressed in the distal convoluted tubule, which aligns with its role in regulating ion transporters in this nephron segment .

What criteria should guide KLHL3 antibody selection for specific applications?

Selection of an appropriate KLHL3 antibody should be based on:

• Target application compatibility: Verify antibody validation for your intended application (WB, IF, IHC, IP)

• Species cross-reactivity: Ensure reactivity with your experimental model organism

• Epitope location: Consider antibodies raised against different regions of KLHL3, particularly if studying specific domains

• Validation evidence: Review published literature using the antibody

• Recognition of variants: Confirm detection of relevant KLHL3 isoforms

For example, the polyclonal antibody described in search result is validated for immunofluorescence labeling (1:100 dilution) and shows reactivity with human, mouse, and rat KLHL3, making it suitable for comparative studies across these species .

How can I validate KLHL3 antibody specificity for my research?

A rigorous validation protocol should include:

• Positive controls: Use tissues or cell lines known to express KLHL3 (e.g., kidney, 293T cells, A549 cells)

• Negative controls: Include KLHL3 knockout samples or tissues with no KLHL3 expression (e.g., muscle tissue)

• Molecular weight verification: Compare observed band sizes with predicted molecular weights, noting that KLHL3 may appear at ~75 kDa despite a calculated MW of 55-64 kDa due to post-translational modifications

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to confirm signal specificity

• RNA interference: Perform siRNA knockdown of KLHL3 to demonstrate signal reduction

• Recombinant protein detection: Test antibody against recombinant KLHL3 protein

What experimental conditions should be optimized when using KLHL3 antibodies in Western blotting?

For optimal Western blot results with KLHL3 antibodies:

• Sample preparation: Use phosphate-buffered solutions with protease inhibitors to prevent degradation

• Protein loading: Load 20-50 μg of total protein per lane

• Dilution optimization: Test antibody dilutions between 1:500-1:2000 for most KLHL3 antibodies

• Incubation conditions: Overnight incubation at 4°C often yields better results than shorter incubations

• Blocking optimization: Test both milk and BSA-based blocking solutions (5%)

• Detection systems: Enhanced chemiluminescence systems with extended exposure may be needed

• Molecular weight marker alignment: Be aware that KLHL3 often appears at higher molecular weight (~75 kDa) than calculated (55-64 kDa)

How can KLHL3 antibodies be used to investigate protein-protein interactions within the CUL3-KLHL3 complex?

To study KLHL3's interactions:

• Co-immunoprecipitation (Co-IP): Use anti-KLHL3 antibodies to pull down KLHL3 and associated proteins (CUL3, WNK kinases). As demonstrated in research, wild-type KLHL3 immunoprecipitated from cell extracts interacts with CUL3 and WNK isoforms . Reciprocal Co-IP can also be performed with anti-CUL3 or anti-WNK antibodies.

• Proximity ligation assay (PLA): Combine KLHL3 antibodies with antibodies against potential binding partners to visualize protein interactions in situ with subcellular resolution.

• Domain-specific antibodies: Use antibodies targeting different KLHL3 domains to map interaction interfaces.

• Mutation studies: Compare immunoprecipitation efficiency between wild-type KLHL3 and disease-causing mutants. Research shows that mutations like R528H in the Kelch domain reduce binding affinity to substrates like claudin-8 by approximately 40% .

• Dimerization analysis: Investigate KLHL3 homodimerization by co-expressing differentially tagged KLHL3 constructs and performing Co-IP, as studies have demonstrated that wild-type KLHL3 can form homodimers and heterodimers with mutant KLHL3 .

What approaches can determine the tissue-specific expression patterns of KLHL3?

To characterize KLHL3 expression patterns:

• Multi-tissue Western blotting: Using validated KLHL3 antibodies to compare expression levels across tissues, with appropriate loading controls.

• Immunohistochemistry (IHC): Apply antibodies at optimal dilutions (1:50-1:100) on tissue sections to visualize cellular and subcellular localization .

• Fluorescent reporter systems: Utilize KLHL3 knockout/reporter knock-in models, as demonstrated with KLHL3 knockout/LacZ knock-in mice that revealed strong expression in brain and kidney, with weaker expression in other organs .

• Single-cell RNA sequencing: Complement protein studies with transcriptomic data to identify cell-specific expression patterns.

• Quantitative immunofluorescence: Combine KLHL3 antibodies with cell-type-specific markers to quantify expression in distinct cell populations.

How can researchers investigate the role of KLHL3 in disease mechanisms beyond hypertension?

Recent research has expanded KLHL3's functional relevance beyond hypertension:

• Metabolic disease models: KLHL3 deficiency in mice showed protection against obesity, insulin resistance, and nonalcoholic fatty liver disease through increased energy expenditure. Experimental approaches included:

  • Comparing body weight and fat mass between control and KLHL3-/- mice under high-fat diet conditions

  • Measuring oxygen consumption and CO₂ production in metabolic cages

  • Analyzing mitochondrial function using the Seahorse assay to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

• Paracellular transport studies: KLHL3 regulates paracellular chloride transport in the kidney. Research approaches included:

  • siRNA knockdown of KLHL3 in kidney cell lines

  • Measuring transepithelial resistance and dilution potential

  • Calculating paracellular ion selectivity using the Goldman–Hodgkin–Katz equation

  • Co-immunoprecipitation studies to investigate KLHL3 interaction with claudin proteins

• Dominant-negative expression systems: Using adenoviral or AAV-mediated expression of dominant-negative KLHL3 to study tissue-specific effects, as demonstrated in hepatocytes where DN-KLHL3 overexpression enhanced mitochondrial function .

How should researchers address molecular weight discrepancies when detecting KLHL3?

The calculated molecular weight of KLHL3 is 55-64 kDa, but it commonly appears at approximately 75 kDa in Western blots . To address this discrepancy:

• Confirm antibody specificity: Validate with positive and negative controls

• Consider post-translational modifications: Phosphorylation, ubiquitination, or glycosylation may increase apparent molecular weight

• Test multiple antibodies: Compare results with antibodies targeting different epitopes

• Include recombinant protein controls: Run purified KLHL3 alongside samples

• Use gradient gels: Improve separation around the region of interest

• Apply reducing/non-reducing conditions: Compare to identify potential disulfide-linked complexes

• Document the discrepancy: When reporting results, note the expected versus observed molecular weight, as this is a known characteristic of KLHL3 detection

What considerations are important when analyzing KLHL3 protein levels in disease models?

When quantifying KLHL3 expression in disease models:

• Establish appropriate controls: Include age-matched, sex-matched controls and consider littermate controls for genetic models

• Account for tissue heterogeneity: KLHL3 expression varies significantly across tissues; even within kidneys, expression is concentrated in specific nephron segments

• Consider protein stability: KLHL3 functions in protein degradation pathways and may have feedback regulation

• Normalize appropriately: Select loading controls stable under your experimental conditions

• Compare multiple detection methods: Combine Western blotting with immunofluorescence or immunohistochemistry

• Assess substrate accumulation: Measure levels of KLHL3 substrates (e.g., WNK kinases) as indirect indicators of KLHL3 activity

• Evaluate functional readouts: Include downstream functional parameters such as ion transporter activity or blood pressure measurements

How can contradictory results between KLHL3 knockout and dominant-negative models be reconciled?

Research shows interesting differences between KLHL3 knockout and dominant-negative approaches:

• Mechanism differences: KLHL3 knockout completely eliminates the protein, while dominant-negative mutations (like R528H) may impair specific functions while preserving others.

• Dimerization effects: KLHL3 can form homodimers and heterodimers with mutant forms, creating complex interaction networks with varying levels of functional impairment .

• Gene dosage effects: Heterozygous KLHL3+/- mice did not show phenotypes, while dominant mutations cause disease in heterozygotes, suggesting a dominant-negative mechanism rather than haploinsufficiency .

• Tissue-specific compensation: Different tissues may have varying compensatory mechanisms for complete versus partial KLHL3 dysfunction.

• Developmental adaptations: Germline knockouts may trigger developmental adaptations absent in post-developmental expression of dominant-negative forms.

• Substrate specificity alterations: Different mutations may affect binding to specific substrates while preserving others, creating distinct molecular profiles.

How are KLHL3 antibodies being utilized to investigate novel metabolic functions?

Emerging research has revealed KLHL3's unexpected role in metabolism regulation:

• Energy expenditure analysis: KLHL3 deficiency increases energy expenditure, with KLHL3-/- mice showing protection against diet-induced obesity. Researchers used metabolic cage experiments to measure oxygen consumption and CO₂ production rates .

• Mitochondrial function assessment: Seahorse assays comparing wild-type KLHL3, dominant-negative KLHL3, and controls in hepatocytes revealed that DN-KLHL3 overexpression enhanced key parameters including basal respiration, ATP production, maximal respiratory capacity, and proton leakage .

• Substrate identification: Proteomic analyses identified 115 proteins involved in mitochondrial function or organization that were significantly changed in livers of KLHL3-/- mice, suggesting new molecular targets for investigation .

• Tissue-specific interventions: AAV-mediated delivery of dominant-negative KLHL3 to the liver ameliorated diet-induced obesity, insulin resistance, and nonalcoholic fatty liver disease, indicating therapeutic potential .

What are the most effective approaches for studying KLHL3 mutations in experimental systems?

To investigate KLHL3 mutations:

• Structure-function analysis: Compare wild-type KLHL3 with disease-causing mutants in biochemical assays. For example, the R528H mutation affects the proper folding of the Kelch domain, reducing binding affinity to substrates like claudin-8 .

• Domain-specific mutations: Generate constructs with mutations in different domains (BTB, BACK, or Kelch domains) to dissect domain-specific functions.

• Dimerization studies: Investigate how mutations affect KLHL3 homodimerization or heterodimerization with wild-type KLHL3, which explains dominant-negative effects in heterozygous patients .

• Substrate ubiquitination assays: Compare ubiquitination efficiency of WNK kinases or other substrates by wild-type versus mutant KLHL3-CUL3 complexes.

• Cell-based functional assays: Measure downstream effects such as ion transporter activity, cell volume regulation, or energy metabolism.

• In vivo models: Generate knock-in mice with specific KLHL3 mutations or use viral delivery of mutant KLHL3 constructs to specific tissues.

• Patient-derived samples: When available, analyze patient samples with KLHL3 mutations to validate experimental findings.