LHX2 Antibody, HRP conjugated

What is LHX2 protein and what cellular functions does it regulate?

LHX2 (LIM homeobox 2), also known as LH2, is a member of the LIM homeodomain family of proteins that functions as a nuclear transcription co-factor. LHX2 can activate or repress gene transcription through direct DNA binding and association with co-activators or co-repressors through its LIM domain. It is involved in numerous critical physiological processes, including cell division, proliferation, specific cell-type differentiation, and cerebral cortex development .

The human LHX2 gene is located on chromosome 9q33.3 and encodes a 389 amino acid protein that functions as part of the LIM homeodomain transcription factor family . LHX2 plays a crucial role in early patterning of the telencephalon in the developing brain, where it helps delineate cortical tissue from the cortical hem, thereby influencing the formation of various brain structures .

What cell lines provide reliable positive controls for LHX2 detection?

According to validation data, reliable positive controls for LHX2 detection in Western blotting include:

These cell lines consistently express detectable levels of LHX2 and serve as appropriate positive controls when validating antibody performance.

What sample preparation methods are essential for optimal LHX2 detection?

For immunohistochemical detection of LHX2, heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 is recommended before commencing with IHC staining protocols . For Western blotting applications, standard sample preparation involves cell lysis using appropriate buffers containing protease inhibitors, followed by protein quantification, denaturation with SDS loading buffer, and heat treatment. The specific protein extraction method should be optimized based on the cellular localization of LHX2 (nuclear protein) and the experimental goals.

How does LHX2 interact with the Sonic Hedgehog (Shh) signaling pathway?

LHX2 functions as a multilevel regulator of Shh signaling by acting cell-autonomously to control the expression of pathway genes required for efficient signaling . ChIP-seq dataset analysis from embryonic retinal progenitor cells (RPCs) identified the Shh pathway as significantly overrepresented among Lhx2 binding sites. LHX2 influences the expression of multiple Shh pathway genes, including Gli1, Ptch1, Ptch2, and Hhip.

Research has demonstrated that "Lhx2 promotes the expression of Cdon and Gas1 to confer signaling competence to RPCs" . When Lhx2 was eliminated in conditional knockout models, Gas1 and Cdon proteins were downregulated, and β-Gal expression from the Gli1-lacZ allele was not detected, supporting Lhx2's role in promoting Shh pathway competence. Despite Lhx2 deficiency, cells retained some competence to signal at the level of Smo (Smoothened), suggesting complex regulatory relationships within the pathway .

What is the role of LHX2 in epigenetic regulation and DNA methylation?

LHX2 gene methylation levels are closely related to cancer development, particularly in cervical cancer. Research has found that the methylation level of LHX2 ranges from 16–56% in cervical cancer patients, with an upward trend of methylation correlating with advancing FIGO stage .

After treatment with 5-aza-2'-deoxycytidine (5-Aza-dC, a demethylating agent) and radiotherapy, the methylation of LHX2 genes in cervical squamous cell carcinoma cells (siHA and C33A lines) decreased, while mRNA and protein expression levels increased . This inverse relationship between methylation status and expression level suggests epigenetic silencing as a key regulatory mechanism for LHX2.

Interestingly, increased LHX2 expression following demethylation was found to accelerate cell invasion and migration while inhibiting apoptosis after radiotherapy treatment . This suggests complex interactions between LHX2 methylation status, expression levels, and functional outcomes in cancer progression.

How does LHX2 interact with other transcription factors in developmental contexts?

LHX2 participates in complex regulatory networks with other transcription factors during development. For instance, research on hippocampal development has revealed an important regulatory relationship between LHX2 and LHX5, another LIM homeobox transcription factor. These genes inhibit each other, creating an essential regulatory axis that ensures appropriate hippocampal development .

Additionally, interactions between LHX2 and nuclear receptor genes COUP-TFI and COUP-TFII have been described. In COUP-TF double-mutant mice, LHX5 expression was reduced at E11.5, followed by enhanced expression of LHX2 at E13.5 and E14.5, suggesting that "COUP-TFI and COUP-TFII, two disease-associated nuclear receptor genes, may cooperate with each other to ensure proper hippocampal morphogenesis by regulating the Lhx5-Lhx2 axis" .

Research has also revealed potential interactions between LHX2 and other transcription factors, including Dmrta2 and Pax6, which together regulate cortical development . These complex interactions highlight LHX2's role as a hub in transcriptional networks governing developmental processes.

What are the optimal dilutions for HRP-conjugated LHX2 antibodies in Western blotting?

A systematic approach to optimization involves:

• Starting with a middle-range dilution (e.g., 1:1000)

• Performing a dilution series (e.g., 1:500, 1:1000, 1:2000)

• Evaluating the balance between specific signal and background

• Selecting the dilution that provides optimal signal-to-noise ratio for your specific samples and detection system

How do polyclonal and monoclonal LHX2 antibodies differ in experimental applications?

Both polyclonal and monoclonal LHX2 antibodies are available for research, each with distinct advantages:

When designing experiments, consider these differences and select the antibody type that best aligns with your specific research requirements regarding sensitivity, specificity, and reproducibility.

What antigen retrieval methods work best for LHX2 immunohistochemistry?

For immunohistochemical detection of LHX2, heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 is recommended . This method has been validated for paraffin-embedded tissue sections and shown to effectively expose LHX2 epitopes for antibody binding. The specific protocol mentioned in the literature involves:

• Deparaffinization of tissue sections

• Heat-mediated antigen retrieval using Tris/EDTA buffer (pH 9.0)

• Blocking of non-specific binding

• Incubation with primary anti-LHX2 antibody (e.g., at 1/500 dilution)

• Detection using an appropriate HRP-conjugated secondary antibody or direct HRP-conjugated primary antibody

• Visualization with a compatible substrate

• Counterstaining (e.g., with Hematoxylin)

This protocol has successfully demonstrated nuclear staining of LHX2 in tissues like mouse olfactory epithelium.

What controls should be included in experiments using LHX2 antibodies?

Multiple controls should be implemented when using LHX2 antibodies to ensure reliable and interpretable results:

• Negative Controls:

  • Secondary antibody-only control: Replace primary antibody with buffer (e.g., PBS) while maintaining all other steps

  • Isotype controls: Use non-specific antibodies of the same isotype (e.g., Rabbit IgG)

  • Negative tissue/cell controls: Use samples known not to express LHX2

• Positive Controls:

  • Cell lines with confirmed LHX2 expression: A549 cells, Jurkat cells, Daudi cells

  • Positive tissue controls: Brain tissue sections, particularly developing cortex and hippocampus where LHX2 is highly expressed

• Technical Controls:

  • Loading controls for Western blotting (e.g., GAPDH, β-actin)

  • Concentration gradient of purified protein (if available)

  • Multiple biological replicates to account for variation

• Functional Controls:

  • LHX2 knockdown or knockout samples (if available)

  • For methylation studies: Samples treated with demethylating agents like 5-Aza-dC

  • For pathway studies: Appropriate pathway inhibitors or activators

How can background signals be reduced when using HRP-conjugated antibodies?

Background signal is a common challenge when using HRP-conjugated antibodies. Several strategies can minimize background and improve signal-to-noise ratio:

• Optimize Antibody Dilution: Use the minimum concentration of antibody that provides a detectable specific signal (typically 1:500-1:2000 for LHX2 antibodies in Western blotting)

• Improve Blocking:

  • Use 3-5% BSA or non-fat dry milk in TBS-T for Western blotting

  • Consider specialized blocking reagents for tissues with high endogenous biotin or peroxidase activity

  • Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

• Reduce Non-specific Binding:

  • Include 0.1-0.3% Triton X-100 or Tween-20 in washing buffers

  • Add 0.2% gelatin or 1% BSA to antibody dilution buffers

  • For tissue sections, pretreat with hydrogen peroxide to quench endogenous peroxidase activity

• Optimize Washing:

  • Increase washing duration and frequency (e.g., 3-5 washes of 5-10 minutes each)

  • Use gentle agitation during washing steps

• Substrate Development:

  • Optimize substrate incubation time

  • Consider using more sensitive/specific HRP substrates for challenging applications

  • For chromogenic detection, monitor development under microscope to prevent overdevelopment

What are common causes of false negative results when using LHX2 antibodies?

False negative results can occur for several reasons when working with LHX2 antibodies:

• Inappropriate Epitope Exposure:

  • Inadequate antigen retrieval (for IHC/ICC): Use heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 as recommended

  • Incomplete protein denaturation (for WB): Ensure adequate SDS and heat treatment

• Antibody-specific Issues:

  • Epitope masking due to protein-protein interactions or post-translational modifications

  • Using antibodies recognizing epitopes absent in your specific splice variants

  • Working with species not validated for cross-reactivity with your antibody

• Technical Factors:

  • Protein degradation during sample preparation

  • Insufficient transfer of proteins to membrane in Western blotting

  • Excessive washing leading to antibody removal

  • Inappropriate blocking agents that mask epitopes

• Biological Factors:

  • Low expression levels of LHX2 in your samples

  • Epigenetic silencing through DNA methylation (LHX2 is subject to methylation-based regulation)

  • Developmental timing (LHX2 expression is temporally regulated during development)

• Detection System Limitations:

  • Expired or degraded HRP substrate

  • Inadequate detection system sensitivity for low-abundance targets

  • Incompatible secondary antibody (if using non-conjugated primary)

What are the implications of altered LHX2 methylation levels in cancer progression?

LHX2 gene methylation has significant implications for cancer development and progression. Research on cervical cancer has revealed:

• Correlation with Disease Stage: "The pyrosequencing results of samples from participants showed an upward trend of the methylation level of LHX2 with the change of FIGO stage" , indicating increasing methylation correlates with cancer progression.

• Expression Regulation: LHX2 methylation inversely correlates with its expression levels. Treatment with demethylating agent 5-Aza-dC decreases LHX2 methylation and increases mRNA and protein expression .

• Functional Consequences: Importantly, increased LHX2 expression following demethylation was found to "accelerate the ability for cell invasion and migration and inhibited the apoptosis of the cell after treatment with radiotherapy" . This suggests a complex role where LHX2 methylation may initially suppress tumor growth but demethylation during treatment might contribute to therapeutic resistance.

• Clinical Relevance: The correlation between methylation levels and clinical staging suggests potential utility as a biomarker. The expression level of LHX2 gene was found to be "significantly correlated with IIB and IIIC stage (P<0.05)" .

• Therapeutic Implications: The paradoxical promotion of invasion, migration, and apoptosis resistance by unmethylated LHX2 suggests caution in applying demethylating agents in certain cancer contexts and highlights the potential of LHX2 as a therapeutic target.

These findings underscore the importance of evaluating both LHX2 methylation status and expression levels when assessing cancer progression and potential treatment responses.

How do changes in LHX2 expression relate to cell invasion and migration ability?

LHX2 expression significantly impacts cell invasion and migration, particularly in cancer contexts:

• Promotion of Invasive Phenotype: Increased LHX2 expression "could accelerate the ability for cell invasion and migration" in cervical cancer cells .

• Anti-apoptotic Effects: LHX2 expression "inhibited the apoptosis of the cell after treatment with radiotherapy" , suggesting a role in treatment resistance.

• Methodological Approaches: These functions have been analyzed using:

  • Overexpression experiments

  • Small interfering RNA (siRNA) knockdown

  • Cell invasion assays

  • Migration assays

  • Quantitative real-time PCR to measure expression levels of migration- and apoptosis-related genes affected by LHX2

• Pathway Involvement: LHX2 promotes growth and metastasis of nasopharyngeal carcinoma specifically by regulating Wnt signaling , suggesting pathway-specific mechanisms underlying its pro-metastatic effects.

• Clinical Correlation: LHX2 "plays a vital role in breast cancer's progression and prognosis" , indicating potential utility as a prognostic marker across multiple cancer types.

When interpreting experimental data showing changes in LHX2 expression, researchers should consider downstream effects on cell motility pathways and apoptotic resistance mechanisms, particularly in cancer models.

How does LHX2 contribute to developmental processes through interaction with signaling pathways?

LHX2 participates in multiple developmental processes through interactions with key signaling pathways:

These interactions highlight LHX2's role as a central node in developmental regulatory networks, integrating multiple signaling inputs to orchestrate proper tissue formation and cellular differentiation.