KLHL21 Antibody

What is KLHL21 and what cellular functions does it regulate?

KLHL21 is a 67 kDa protein belonging to the Kelch-like family, containing three characteristic domains: BTB (bric-a-brac, tramtrack, broad complex), BACK domain, and Kelch domain. It functions primarily as:

• A substrate-specific adapter in E3 ubiquitin ligase complexes that mediates ubiquitination and proteasomal degradation of target proteins

• A negative regulator of TNFα-activated NF-κB signaling by targeting IKKβ

• A critical component in mitotic regulation, particularly during cytokinesis by controlling chromosomal passenger complex translocation

KLHL21 binds directly to the kinase domain of IKKβ via its Kelch domains, inhibiting IKKβ activation independently of its E3 ligase activity .

What applications can KLHL21 antibodies be used for in experimental research?

KLHL21 antibodies have been validated for multiple experimental applications:

Note: Optimal dilutions should be determined experimentally based on specific antibody characteristics and sample types .

How should KLHL21 antibodies be stored for maximum stability and efficacy?

Proper storage is critical for maintaining antibody functionality:

• Store at -20°C in aliquots to minimize freeze/thaw cycles

• Most KLHL21 antibodies remain stable for one year when stored correctly

• Monoclonal antibodies are typically supplied in PBS or ascitic fluid containing preservatives like sodium azide (0.02-0.03%)

• Polyclonal antibodies are often provided in PBS with 50% glycerol at pH 7.3

• For long-term storage, some suppliers recommend -80°C for conjugation-ready formats

Repeated freeze/thaw cycles should be avoided as they can compromise antibody performance .

How does KLHL21 regulate the NF-κB signaling pathway in inflammatory processes?

KLHL21 functions as a negative regulator of the NF-κB pathway through specific molecular mechanisms:

• KLHL21 binds directly to the kinase domain (KD) of IKKβ via its Kelch domains

• Upon inflammatory stimuli like TNFα or LPS, KLHL21 expression is rapidly downregulated in macrophages and dendritic cells, with mRNA levels reaching <10% within 4 hours

• KLHL21 protein expression follows a similar temporal pattern, decreasing dramatically within 1 hour of LPS treatment

• Mechanistically, KLHL21:

  • Inhibits TNFα-stimulated IKKβ activation

  • Suppresses phosphorylation of IKKα/β (Ser-177)

  • Prevents degradation of IκBα

  • Functions independently of its E3 ubiquitin ligase activity

Importantly, KLHL21 does not disrupt the interaction between IKKβ and other signaling components like TAK1, TRAF2, or IκBα, suggesting it may function by sequestering the kinase domain of IKKβ from potential activators .

What is the role of KLHL21 in mitosis and how does it interact with Aurora B?

KLHL21 plays a critical role in cell division through the following mechanisms:

• Forms a functional E3 ubiquitin ligase complex with Cullin3 (Cul3)

• Targets Aurora B for ubiquitination, which is essential for:

  • Translocation of the chromosomal passenger complex (CPC) from chromosomes to the spindle midzone during anaphase

  • Proper completion of cytokinesis

In KLHL21-depleted cells:

• Aurora B fails to relocalize properly during anaphase

• Cells show defects in cytokinesis, where the cleavage furrow ingresses but then regresses

• The kinesin-6 family member Mklp2 is not efficiently recruited to the midzone

Biochemically, KLHL21 directly binds to Aurora B as demonstrated by co-immunoprecipitation experiments, and this interaction is critical for the spatial and temporal control of Aurora B during mitosis .

How is KLHL21 implicated in cancer progression and potential therapeutic targeting?

Research has identified KLHL21 as a potential oncogenic factor:

• In hepatocellular carcinoma (HCC):

  • KLHL21 expression is significantly upregulated in primary HCC tissues compared to adjacent non-cancerous liver tissues

  • siRNA-mediated knockdown of KLHL21 in highly metastatic HCC cell lines (MHCC97H and HCC-LM3) resulted in:

    • Inhibited cell proliferation over 5 days

    • Reduced cell migration (~40% reduction in MHCC97H cells and ~30% reduction in HCC-LM3 cells)

    • Decreased invasion capacity

• Molecular targeting of KLHL21:

  • Meta-analysis of expression data from 1525 HCC patients identified KLHL21 as having strong clinical significance

  • KLHL21 was identified as the most promising gene among several candidates for potential use as a bioclinical marker

  • The protein's roles in cell cycle control through Aurora B regulation make it a candidate for targeted therapy development

These findings suggest KLHL21 could serve as both a prognostic biomarker and potential therapeutic target in HCC and possibly other cancer types .

What are the key differences between using monoclonal versus polyclonal KLHL21 antibodies?

Choosing between these formats depends on experimental needs - monoclonals offer higher reproducibility while polyclonals may provide enhanced detection sensitivity across diverse applications .

How do inflammatory stimuli affect KLHL21 expression and what are the implications for experimental design?

KLHL21 expression is dynamically regulated during inflammation:

• Temporal expression pattern:

  • mRNA levels drop rapidly after LPS treatment, reaching minimal levels (~10% of baseline) by 4 hours, then gradually increase to ~70% of baseline by 24 hours

  • Protein abundance decreases dramatically within 1 hour of LPS exposure, followed by a steady increase

• Tissue/cell specificity:

  • Downregulation observed in:

    • RAW264.7 monocytic macrophage cells

    • Bone marrow-derived macrophages

    • LPS-treated dendritic cells

    • Peripheral blood-derived monocytes

    • CpG oligonucleotide-treated bone marrow-derived dendritic cells

    • Kaposi sarcoma-associated herpesvirus-infected primary human dermal endothelial cells

• Experimental implications:

  • Time point selection is critical when studying KLHL21 during inflammatory responses

  • Researchers should consider the rapid downregulation when designing experiments

  • Cell-type specific responses should be accounted for

  • Correlation of protein and mRNA levels cannot be assumed due to their different degradation kinetics

This dynamic regulation suggests KLHL21 downregulation may be a general consequence of inflammatory responses, with important implications for experimental timing and interpretation .

What are recommended protocols for validating KLHL21 knockdown experiments?

When conducting KLHL21 knockdown studies, multiple validation approaches should be employed:

Validation at mRNA level:

• qRT-PCR using primers specific to KLHL21

• Reference genes should be carefully selected based on cell type and experimental conditions

Validation at protein level:

• Western blot using validated anti-KLHL21 antibodies (1:500-1:1000 dilution)

• Expected band size approximately 66-67 kDa

Functional validation:

• For NF-κB pathway studies: measure phosphorylation status of IKKα/β (Ser-177) and IκBα, as well as IκBα degradation following TNFα treatment

• For mitosis studies: examine Aurora B localization during anaphase using immunofluorescence

• For cancer-related studies: proliferation assays and Transwell migration/invasion assays

Rescue experiments:

• Co-transfection of siRNA targeting endogenous KLHL21 with expression constructs containing siRNA-resistant KLHL21 coding sequences

• Wild-type and functional mutants (e.g., E3 ligase activity-deficient mutants) can be used to dissect mechanism

As demonstrated in published research, proper controls (including knockdown control cells) are essential for interpreting results accurately .

How can researchers differentiate between KLHL21 and other KLHL family members in their experiments?

The KLHL family contains multiple members with similar domain structures, necessitating careful experimental design:

Antibody specificity validation:

• Western blot against recombinant KLHL proteins to confirm absence of cross-reactivity

• Immunoprecipitation followed by mass spectrometry to identify potential cross-reactive proteins

• Using KLHL21 knockout/knockdown controls to confirm signal specificity

Functional distinction approaches:

• KLHL21 specifically:

  • Binds to the kinase domain of IKKβ via its Kelch domains

  • Targets Aurora B for ubiquitination during mitosis

  • Does not require E3 ligase activity for IKKβ inhibition, unlike some other family members

Comparative analysis:

• Other KLHL family members have distinct targets:

  • KLHL22 targets PLK1 and DEPDC5

  • KLHL20 targets ULK1, PML, and DAPK

  • KLHL25 targets 4E-BP1 and ACLY

Domain-specific analysis:

• The BTB domain mutations (D114A/L115A/Q117A) can distinguish KLHL21's CUL3-dependent versus independent functions

• The Kelch domains are critical for substrate recognition and can be used to differentiate family members

Understanding these distinctions is crucial when investigating specific KLHL family members in complex biological systems .

What protein-protein interaction techniques are most suitable for studying KLHL21 complexes?

Several complementary techniques have been successfully applied to study KLHL21 interactions:

Co-immunoprecipitation (Co-IP):

• Effectively demonstrated interaction between KLHL21 and:

  • IKKβ, IKKα, and NEMO (components of the IKK complex)

  • Aurora B (part of the chromosomal passenger complex)

• Both overexpression systems and endogenous proteins have been used successfully

• Typical protocol involves using 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

GST pull-down assays:

• Confirmed direct interaction between KLHL21 and IKKβ

• Useful for mapping specific interaction domains

Structure-function analysis:

• Deletion mutants identified that:

  • The Kelch domains of KLHL21 are required for interaction with IKKβ

  • The kinase domain of IKKβ is critical for association with KLHL21

  • BTB domain mutations (D114A/L115A/Q117A) disrupt CUL3 binding but not IKKβ interaction

Immunofluorescence co-localization:

• Demonstrated KLHL21 and IKKβ colocalization in HeLa cells

• Used to study Aurora B localization during mitosis

These complementary approaches provide robust evidence for specific protein-protein interactions and help elucidate their functional significance .

What are common issues when using KLHL21 antibodies and how can they be resolved?

For optimal results with anti-KLHL21 antibodies, include appropriate positive controls (A431 or HeLa cells) and negative controls (KLHL21 knockdown samples) in experiments .

How should researchers optimize experimental conditions when studying KLHL21 in different cellular contexts?

Optimization strategies should address the unique characteristics of KLHL21 across different experimental systems:

Cell type considerations:

• Expression levels vary significantly across cell types; perform baseline expression analysis

• KLHL21 is highly expressed in hepatocellular carcinoma cells (MHCC97H, HCC-LM3)

• RAW264.7 and bone marrow-derived macrophages show dynamic regulation during inflammation

Stimulation conditions:

• For inflammatory studies:

  • TNFα treatment: 10 min is sufficient to observe attenuation of KLHL21-IKKβ interaction

  • LPS treatment: expression nadir occurs around 4 hours post-treatment

  • Consider temporal dynamics when designing experiments

Subcellular localization:

• During mitosis, KLHL21 localization changes dynamically:

  • In metaphase: primarily chromosomal

  • In anaphase: relocalization to the spindle midzone

• Use cell synchronization techniques for mitosis studies

Functional readouts:

• NF-κB pathway: measure IKKβ phosphorylation status and IκBα degradation

• Cell cycle: examine Aurora B localization by immunofluorescence

• Cancer phenotypes: cell proliferation, migration, and invasion assays

Technical optimizations:

• Western blot: 1:500-1:1000 dilution for most anti-KLHL21 antibodies

• IHC: antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0)

• IP: 0.5-4.0 μg antibody per 1.0-3.0 mg protein lysate

These targeted optimizations will enhance experimental robustness across different research contexts .

What are emerging applications of KLHL21 antibodies in cancer research?

Recent studies have expanded the potential applications of KLHL21 antibodies in cancer research:

• Biomarker development:

  • KLHL21 has been identified as a promising bioclinical marker in hepatocellular carcinoma

  • Meta-analysis of 1525 HCC patients showed significant clinical correlation with KLHL21 expression

  • Antibody-based assays could potentially stratify patients for targeted therapies

• Therapeutic target validation:

  • KLHL21 knockdown significantly inhibits proliferation, migration, and invasion in highly metastatic HCC cell lines

  • Antibodies are being used to validate KLHL21 as a potential therapeutic target

• Mechanistic investigations:

  • KLHL21's dual roles in mitosis regulation (via Aurora B) and inflammatory signaling (via IKKβ) position it at the intersection of cancer-related pathways

  • Multi-parameter immunofluorescence using KLHL21 antibodies helps map protein interaction networks

• Pathway crosstalk:

  • KLHL21 belongs to the broader KLHL family with diverse roles in cancer

  • Comparative studies using antibodies against multiple KLHL members help elucidate functional redundancy and specialization

As research progresses, KLHL21 antibodies will likely become increasingly important tools for understanding cancer biology and developing potential therapeutics .

How do post-translational modifications affect KLHL21 function and antibody recognition?

Understanding post-translational modifications (PTMs) of KLHL21 is crucial for both experimental design and interpretation:

• Ubiquitination:

  • KLHL21 itself can be ubiquitinated, potentially as a regulatory mechanism

  • KLHL21 functions as part of an E3 ubiquitin ligase complex with CUL3

  • Antibodies recognizing ubiquitinated forms may be needed for specific applications

• Phosphorylation:

  • During inflammation, rapid downregulation of KLHL21 suggests possible regulation by phosphorylation

  • Phospho-specific antibodies could help elucidate these regulatory mechanisms

• Antibody epitope considerations:

  • Most commercial KLHL21 antibodies target recombinant fragments or fusion proteins

  • Epitope location relative to PTM sites can affect recognition

  • For studies focused on specific PTMs, validation with appropriate controls is essential

• Functional implications:

  • BTB domain mutations (D114A/L115A/Q117A) disrupt CUL3 binding but not IKKβ interaction

  • Different PTMs may selectively affect interactions with specific partners

When designing experiments, researchers should consider how PTMs might affect antibody recognition and protein function in different cellular contexts .