KLHL2 Antibody

What is KLHL2 protein and what are its key structural domains?

KLHL2 (kelch-like 2, also known as Mayven) is a substrate-specific adapter protein that functions within BCR (BTB-CUL3-RBX1) E3 ubiquitin ligase complexes. The protein contains distinct structural domains:

• N-terminal SH3-binding domain

• POZ (BTB/Pox virus and Zinc finger) domain

• BACK domain (required for Cullin3 interaction)

• C-terminal region containing 6 tandem kelch repeats (approximately 50 amino acids each)

The kelch domain specifically mediates substrate recognition and binding, as demonstrated in studies where deletion of this domain abolished KLHL2's ability to interact with target proteins like ARHGEF7 . The full-length protein has a molecular weight of approximately 66 kDa, which can be observed in Western blot applications .

What are the primary applications for KLHL2 antibodies in experimental research?

KLHL2 antibodies are employed across multiple experimental techniques:

The antibody has demonstrated positive Western blot detection in various cell types including K-562 cells, human brain tissue, MCF-7 cells, and SH-SY5Y cells . For immunofluorescence applications, successful detection has been reported in MCF-7 cells .

What tissues and cell types express KLHL2 protein?

KLHL2 demonstrates differential expression across tissues:

• Vascular system: Highly expressed in smooth muscle cells of mouse aorta

• Nervous system: Detected in human brain tissue, with significant expression in primary rat hippocampal neurons (particularly in cell bodies and neurite processes)

• Cellular distribution: In astrocytoma/glioblastoma cells, KLHL2 colocalizes with actin filaments in cytoskeleton remodeling regions, stress fibers, and cortical actin-rich cell margins

RT-PCR analysis has confirmed KLHL2 mRNA expression in mouse aorta, kidney, and vascular smooth muscle cells (MOVAS) . This tissue-specific expression pattern suggests specialized functions of KLHL2 in different physiological contexts.

How can I distinguish between KLHL2 and other KLHL family members in my experiments?

Distinguishing between KLHL family members requires careful experimental design:

• Antibody selection: Validate KLHL2 antibodies by Western blot using control samples. For example, researchers verified KLHL2 antibody specificity by demonstrating that KLHL2 knockdown by siRNA decreased both bands detected by the anti-KLHL2/KLHL3 antibody .

• Molecular weight discrimination: While KLHL2 has an observed molecular weight of 66 kDa, other family members like KLHL12 have distinct molecular weights (approximately 62 kDa) .

• RNA interference validation: siRNA knockdown of KLHL2 followed by Western blot can confirm antibody specificity, as demonstrated in vascular smooth muscle cell studies .

• Protein interaction profiles: Different KLHL proteins interact with distinct substrates. For example, KLHL22 specifically interacts with PD-1, while KLHL2 does not show this interaction . Similarly, KLHL2 demonstrates specific interactions with ARHGEF7, which other family members may not share .

What are the known protein substrates of KLHL2 and how can I study KLHL2-mediated protein degradation?

KLHL2 mediates the ubiquitination and degradation of several substrates:

To study KLHL2-mediated protein degradation:

• Ubiquitination assays: Co-express KLHL2 with the substrate of interest along with tagged ubiquitin, followed by immunoprecipitation and Western blot to detect ubiquitinated species.

• Proteasome inhibition: Treat cells with proteasome inhibitors (MG132, Bortezomib) or neddylation inhibitors (MLN4924) to block degradation and observe substrate accumulation. This approach has been successfully used to demonstrate KLHL2-mediated degradation of ARHGEF7 .

• KLHL2 domain analysis: Generate KLHL2 deletion mutants (particularly targeting the kelch domain) to identify regions required for substrate interaction and degradation .

How does regulation of KLHL2 protein levels impact its function in signaling pathways?

KLHL2 protein levels are dynamically regulated:

• Angiotensin II (AngII) regulation: AngII rapidly decreases KLHL2 protein abundance in vascular smooth muscle cells through autophagy-mediated degradation, leading to increased WNK3 levels and downstream activation .

• p62-mediated selective autophagy: Unlike conventional proteasomal degradation of most proteins, KLHL2 is degraded through a selective autophagy pathway mediated by p62/sequestosome1. Experiments with autophagy inhibitors like chloroquine and bafilomycin A prevented AngII-induced KLHL2 degradation .

• Physiological implications: The dynamic regulation of KLHL2 allows for rapid modulation of downstream pathways:

  • In vascular smooth muscle cells, KLHL2 degradation leads to WNK3-SPAK-NKCC1 pathway activation

  • In high potassium concentration environments, KLHL2-mediated regulation of WNK3 is critical for vascular smooth muscle cell responses

What experimental controls should I include when performing immunoprecipitation with KLHL2 antibodies?

For robust co-immunoprecipitation experiments with KLHL2 antibodies:

• Input control: Include a sample of the total lysate before immunoprecipitation (5-10%)

• Negative controls:

  • IgG control using the same species as the KLHL2 antibody

  • Immunoprecipitation in cells with KLHL2 knockdown

  • Use of KLHL2 mutants lacking interaction domains (e.g., KLHL2-M3 mutant lacking the kelch domain)

• Validation of interactions: Perform reverse co-IP where the putative interacting partner is immunoprecipitated and KLHL2 is detected in the precipitate

• Domain mapping: Use deletion mutants to identify specific domains required for protein interactions. For example, studies with ARHGEF7 showed that the kelch domain of KLHL2 is essential for this interaction .

How can I investigate KLHL2 expression and function in disease models, particularly cancer?

KLHL2 has been implicated in cancer pathophysiology:

• Expression analysis in tumors:

  • Immunohistochemistry (IHC) on tissue microarrays can evaluate KLHL2 expression across tumor and adjacent normal tissues

  • Studies have observed negative correlation between KLHL2 and ARHGEF7 expression in clear cell renal cell carcinoma (ccRCC)

• Functional studies:

  • siRNA-mediated KLHL2 knockdown in cancer cell lines followed by proliferation, migration, and invasion assays

  • Overexpression of KLHL2 and its effect on tumor-associated substrates

• Mechanistic investigations:

  • In ccRCC, KLHL2 promotes ARHGEF7 degradation via the ubiquitin-proteasome pathway

  • In AML, KLHL2 targets UCK1 for degradation, potentially contributing to 5'-azacytidine resistance

• Scoring systems for IHC:

  • Intensity scoring (0-3 scale: none, weak, intermediate, strong)

  • Proportional scoring based on percentage of positive tumor cells

  • Assessment of at least 100 tumor cells across multiple regions

How can I optimize Western blot conditions for detecting KLHL2 protein?

For optimal Western blot results with KLHL2 antibodies:

• Sample preparation:

  • Use RIPA or NP-40 lysis buffers containing protease inhibitors

  • Include phosphatase inhibitors if studying KLHL2 phosphorylation

  • For detecting autophagy-mediated KLHL2 degradation, include bafilomycin A or chloroquine treatment groups

• Gel percentage and running conditions:

  • Use 7.5-10% SDS-PAGE gels for optimal separation of the 66 kDa KLHL2 protein

  • Run at constant voltage (100-120V) for best resolution

• Antibody dilution optimization:

  • Start with manufacturer's recommended dilution range (1:5000-1:50000)

  • Perform titration experiments to determine optimal concentration

  • Include positive control samples (K-562 cells, human brain tissue, MCF-7 cells)

• Multiple band interpretation:

  • KLHL2 may appear as double bands in Western blot

  • The lower band typically corresponds to the major KLHL2 form that matches cloned KLHL2 cDNA

  • The upper band's nature is still under investigation but is not due to phosphorylation, glycosylation, or neddylation

What strategies can help distinguish KLHL2-mediated effects from other E3 ligase pathways?

To specifically attribute observed effects to KLHL2:

• Genetic approaches:

  • Generate KLHL2 knockout models using CRISPR-Cas9

  • Use multiple independent siRNAs targeting KLHL2 to minimize off-target effects

  • Rescue experiments with siRNA-resistant KLHL2 constructs

• Pathway-specific inhibitors:

  • MLN4924 (neddylation inhibitor) blocks CUL3-based E3 ligase activity

  • Compare effects with inhibitors of other degradation pathways

• Domain-specific mutants:

  • Use KLHL2 mutants lacking specific domains (BTB, BACK, or kelch) to determine domain requirements

  • Studies have shown that mutants lacking the kelch domain (M3 mutant) lose the ability to interact with and degrade substrates like ARHGEF7

• Substrate stabilization analysis:

  • Monitor multiple known KLHL2 substrates simultaneously

  • Compare degradation kinetics across substrates to identify common patterns

How can I study the dynamics of KLHL2-mediated protein regulation in living cells?

Advanced techniques for investigating KLHL2 dynamics:

• Live-cell imaging approaches:

  • Generate fluorescently tagged KLHL2 constructs (e.g., GFP-KLHL2)

  • Use fluorescence recovery after photobleaching (FRAP) to measure mobility and binding kinetics

  • Implement fluorescence resonance energy transfer (FRET) assays between KLHL2 and substrates

• Real-time degradation monitoring:

  • Express destabilized fluorescent protein fusions with KLHL2 substrates

  • Quantify fluorescence changes over time following pathway stimulation

  • For example, monitor WNK3 levels after AngII treatment in vascular smooth muscle cells

• Protein-fragment complementation assays:

  • Split luciferase or fluorescent protein complementation between KLHL2 and substrate

  • Provides direct measurement of protein-protein interactions in live cells

• Temporal analysis of modifications:

  • Pulse-chase experiments with [35S]-labeled amino acids to track protein synthesis and degradation

  • Quantitative ubiquitination assays at different time points after stimulus

  • This approach has been used to study incompletely glycosylated PD-1 degradation by KLHL22

How do KLHL2 regulatory mechanisms differ across tissue types?

Tissue-specific regulatory patterns of KLHL2:

• Vascular smooth muscle cells:

  • KLHL2 is regulated by AngII through p62-mediated selective autophagy

  • High potassium concentration regulates the WNK3-SPAK-NKCC1 phosphorylation cascade via KLHL2

• Neural tissue:

  • In primary rat hippocampal neurons, KLHL2 is highly expressed in cell bodies and neurite processes

  • KLHL2 colocalizes with actin filaments in cytoskeleton remodeling regions in astrocytoma/glioblastoma cells

• Cancer cells:

  • KLHL2 expression negatively correlates with ARHGEF7 in ccRCC

  • KLHL2 mediates UCK1 degradation in acute myeloid leukemia, potentially contributing to drug resistance

Research methods to study tissue-specific regulation:

• Tissue-specific conditional knockout models

• Primary cell cultures from different tissues

• Comparison of KLHL2 interactome across cell types using immunoprecipitation coupled with mass spectrometry

What is the relationship between KLHL2 and other KLHL family members in coordinating cellular responses?

Understanding KLHL family cooperation and specialization:

• Substrate specificity:

  • Different KLHL proteins target distinct substrates: KLHL2 targets WNK kinases and ARHGEF7, while KLHL22 targets PD-1

  • Some substrates may be regulated by multiple KLHL proteins in different contexts

• Structural similarities and differences:

  • All KLHL proteins contain BTB/POZ domains and kelch repeats

  • Sequence variations in kelch domains confer substrate specificity

  • The Kelch domain of KLHL2 specifically mediates binding to ARHGEF7, as demonstrated by deletion mutant studies

• Regulatory mechanisms:

  • KLHL2 is regulated by autophagy

  • Other KLHL members may undergo different regulatory processes

  • Potential cross-regulation among KLHL family members

Research approaches:

• Comparative proteomic analysis of different KLHL protein complexes

• Generation of cell lines with multiple KLHL gene knockouts

• Analysis of compensatory mechanisms following single KLHL gene knockout

Can KLHL2 function be manipulated to reverse pathological processes in disease models?

Therapeutic potential of targeting KLHL2 pathways:

• Cancer applications:

  • In ccRCC, KLHL2 depletion could potentially reverse ARHGEF7-mediated tumor progression

  • In AML, targeting KLHL2-mediated UCK1 degradation might overcome 5'-azacytidine resistance

• Vascular disease applications:

  • Modulation of KLHL2-WNK3 pathway could affect vascular tone and blood pressure regulation

  • AngII-induced KLHL2 degradation represents a potential intervention point in hypertension

• Experimental approaches for therapeutic development:

  • Small molecule screens to identify compounds that stabilize KLHL2

  • Peptide inhibitors of KLHL2-substrate interactions

  • RNA interference or antisense oligonucleotide strategies for targeted KLHL2 modulation

Research models for testing interventions:

• Patient-derived xenografts for cancer studies

• Genetically modified mouse models of vascular disease

• Cell-based high-throughput screens for compound identification