LIMK2 Antibody

Which tissue types express high levels of LIMK2 for positive controls?

According to expression profiling data, the placenta demonstrates the highest LIMK2 expression levels, making placental tissue extracts excellent positive controls . Moderate LIMK2 expression is also found in liver, lung, kidney, and pancreas tissues . For cell lines, NIH/3T3 cells have been validated for positive Western blot detection . Interestingly, LIMK2 isoform distribution varies by tissue - LIMK2a is more abundant in liver, colon, stomach, and spleen, while LIMK2b predominates in brain, kidney, and placenta .

How does LIMK2 expression correlate with immune cell infiltration in cancer microenvironments?

Research has revealed complex relationships between LIMK2 expression and tumor immune infiltration. In lung squamous cell carcinoma (LUSC), LIMK2 expression is significantly negatively correlated with B cells, CD8+ T cells, and dendritic cells, suggesting an immunosuppressive role . LIMK2 copy number variation also significantly impacts immune cell infiltration levels in LUSC tumors . More specifically, LIMK2 expression has been shown to negatively correlate with immune checkpoint markers including PDCD1 (PD1), CD274 (PDL1), and CTLA-4, indicating that LIMK2 may improve LUSC patient survival by suppressing immune responses in the tumor microenvironment . For researchers investigating tumor immunology, these correlations suggest LIMK2 as a potential target for modulating tumor immune responses.

Which experimental models are most effective for studying LIMK2 inhibition in cancer progression?

For metastasis research, subcutaneous injection of MDA-MB-231 cells labeled with firefly luciferase in mice has proven effective for studying LIMK2 inhibition using compounds like LX7101 . This model enables monitoring of both primary tumor growth and spontaneous metastatic progression to the lungs via bioluminescence imaging . For castration-resistant prostate cancer (CRPC) models, C4-2 xenografts in castrated nude mice with inducible LIMK2 knockdown systems (Tet-pLKO.1-LIMK2 shRNA or LT3GEPIR-LIMK2 shRNA) have demonstrated dramatic tumor regression effects, highlighting LIMK2's therapeutic potential . Three-dimensional invasion assays using TNBC cell lines (MDA-MB-231 and BT-549) with genetic LIMK2 knockdown provide another valuable approach for evaluating LIMK2's role in maintaining metastatic attributes .

What are the functional differences between LIMK2 isoforms and how should they be addressed experimentally?

LIMK2 exists in multiple isoforms, with LIMK2a and LIMK2b being the predominant variants with tissue-specific expression patterns . These isoforms demonstrate differential expression: LIMK2a is more abundant in liver, colon, stomach, and spleen, while LIMK2b predominates in brain, kidney, and placenta . In adult lung tissue, both isoforms are expressed at approximately equal levels . When designing experiments, researchers should consider:

• Antibody selection: Ensure the antibody recognizes your isoform of interest or multiple isoforms if comprehensive detection is desired

• Tissue selection: Match experimental tissue types with known isoform expression patterns

• Primer design: For RT-PCR or qPCR experiments, design primers that can distinguish between isoforms

• Functional studies: Consider that isoforms may have different subcellular localizations or substrate preferences

What are the optimal conditions for phospho-specific detection of activated LIMK2?

For researchers investigating LIMK2 activation status, phospho-specific antibodies targeting phosphorylated T505 (such as ab38499) are essential . This site is critical for LIMK2 activation. For optimal phospho-LIMK2 detection:

• Sample preparation: Maintain phosphorylation status by including phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) in lysis buffers

• Blocking conditions: Use 5% BSA rather than milk for blocking and antibody dilution, as milk contains phosphatases that may reduce signal

• Validation approach: Always include both phospho-LIMK2 and total LIMK2 antibodies on parallel blots to calculate the phosphorylation ratio

• Controls: Include samples treated with phosphatase inhibitors (calyculin A or okadaic acid) as positive controls

• Stimulation: For maximal phospho-LIMK2 signal, stimulate cells with ROCK activators or Rho pathway stimulants before lysis

What is the recommended protocol for immunoprecipitation of LIMK2 to study protein-protein interactions?

For immunoprecipitation of LIMK2, the following methodological approach is recommended:

• Antibody selection: Use antibodies validated for immunoprecipitation applications, such as mouse monoclonal LIMK-2 (A-12) antibody

• Starting material: Use 500-1000 μg of total protein lysate per IP reaction

• Pre-clearing: Pre-clear lysates with protein G agarose to reduce non-specific binding

• Antibody binding: Incubate cleared lysates with 2-5 μg of LIMK2 antibody overnight at 4°C

• Capture: Add protein G agarose beads for 2-4 hours at 4°C

• Washing: Perform stringent washes (at least 4-5) with decreasing salt concentrations

• Elution: Elute immune complexes using SDS sample buffer heated to 95°C for 5 minutes

• Analysis: Analyze by SDS-PAGE followed by Western blotting for LIMK2 and potential interaction partners

For co-immunoprecipitation studies investigating LIMK2 interactions with actin cytoskeleton components or potential novel substrates like TWIST1, this approach allows detection of protein complexes while maintaining native conformation.

How should immunohistochemical detection of LIMK2 be optimized for FFPE tissue specimens?

For optimal immunohistochemical detection of LIMK2 in formalin-fixed paraffin-embedded (FFPE) tissues:

• Antigen retrieval: Use TE buffer at pH 9.0 for optimal epitope exposure, though citrate buffer at pH 6.0 can serve as an alternative

• Antibody dilution: Start with a dilution range of 1:50-1:500, then optimize based on signal-to-noise ratio

• Positive control selection: Include human stomach tissue as a validated positive control

• Blocking: Use 3-5% normal serum from the species of secondary antibody origin

• Incubation conditions: Incubate primary antibody overnight at 4°C for improved sensitivity

• Detection system: HRP-polymer detection systems generally provide better sensitivity than traditional ABC methods

• Counterstaining: Use light hematoxylin counterstaining to avoid masking specific signals

This methodology has been successfully applied in studies examining LIMK2 expression in tumor tissue microarrays, including those analyzing TNBC and LUSC tissues .

What are the common causes of non-specific bands when using LIMK2 antibodies in Western blot?

When encountering non-specific bands in LIMK2 Western blots, consider these potential causes and solutions:

For monoclonal antibodies like EP969Y (ab45165) that target the C-terminal region of LIMK2, specificity is generally higher with fewer non-specific bands .

How can I validate LIMK2 knockdown efficiency for functional studies?

To rigorously validate LIMK2 knockdown efficiency for functional studies:

• Protein level validation:

  • Western blot using validated LIMK2 antibodies (1:500-1:1000 dilution)

  • Quantify LIMK2 reduction relative to loading controls (β-actin, GAPDH)

  • Check downstream effects on cofilin phosphorylation status

• mRNA level validation:

  • qRT-PCR with LIMK2-specific primers

  • Design primers that detect all relevant isoforms

  • Calculate fold-change using 2^(-ΔΔCT) method

• Functional validation:

  • Actin cytoskeleton visualization using phalloidin staining

  • Cofilin phosphorylation using phospho-cofilin antibodies

  • Rescue experiments by re-expressing shRNA-resistant LIMK2

• Controls:

  • Include non-targeting shRNA controls

  • Use multiple independent shRNA constructs targeting different LIMK2 regions

  • For inducible systems, compare induced vs. non-induced conditions

This comprehensive validation approach was successfully implemented in studies investigating LIMK2's role in TNBC metastasis and CRPC tumorigenesis .

How does LIMK2 targeting compare to other cytoskeletal regulators as therapeutic strategies in cancer?

LIMK2 offers several distinct advantages as a therapeutic target compared to other cytoskeletal regulators:

• Disease specificity: LIMK2 is upregulated specifically in response to androgen deprivation therapy in prostate cancer, making it particularly relevant for castration-resistant prostate cancer (CRPC)

• Metastasis-specific targeting: In TNBC models, LIMK2 inhibition using LX7101 selectively reduced metastatic spread without affecting primary tumor growth, suggesting potential for metastasis-specific interventions

• Mechanism of action: Unlike direct cytoskeletal inhibitors that can cause systemic toxicity, LIMK2 modulates cytoskeletal dynamics through regulatory pathways:

  • Phosphorylates and inactivates cofilin, an actin-depolymerizing factor

  • Regulates microtubule organization through gamma-tubulin interaction

  • Modulates TWIST1 activity through direct phosphorylation

• Reversibility: Inducible knockdown of LIMK2 in castrated mice demonstrated full reversal of CRPC tumorigenesis, highlighting its potential for targeted therapeutic intervention

Compared to other cytoskeletal regulators like Rho/ROCK pathway inhibitors, LIMK2 targeting may provide more selective effects on pathological processes while minimizing disruption of normal cellular functions.

What are the implications of LIMK2's correlation with immune markers for cancer immunotherapy research?

The negative correlation between LIMK2 expression and immune checkpoint markers (PDCD1/PD1, CD274/PDL1, and CTLA-4) in lung squamous cell carcinoma has significant implications for cancer immunotherapy research :

• Potential combination approaches: LIMK2 inhibitors could potentially enhance the efficacy of immune checkpoint inhibitors by modulating the immunosuppressive tumor microenvironment

• Biomarker development: LIMK2 expression levels might serve as predictive biomarkers for immunotherapy response, particularly in LUSC

• Immune subset targeting: Given LIMK2's negative correlation with specific immune cell populations (B cells, dendritic cells, and CD8+ T cells), therapeutic strategies might be tailored to enhance these populations specifically

• Resistance mechanisms: High LIMK2 expression might represent a mechanism of resistance to immune checkpoint inhibitors that could be therapeutically targeted

• Patient stratification: Tumor LIMK2 expression profiling could help stratify patients for appropriate immunotherapy approaches

These findings suggest that incorporating LIMK2 expression analysis into immunotherapy research protocols may provide valuable insights into treatment response and resistance mechanisms.

How can researchers effectively utilize LIMK2 antibodies in high-throughput screening or automated imaging platforms?

For implementing LIMK2 antibodies in high-throughput screening or automated imaging platforms:

• Antibody selection criteria:

  • Choose antibodies with validated specificity in immunofluorescence applications

  • Select fluorophore-conjugated versions when available (FITC or Alexa Fluor conjugates)

  • Consider recombinant monoclonal antibodies for batch-to-batch consistency

• Optimization parameters:

  • Determine optimal fixation methods (4% PFA vs. methanol)

  • Establish ideal permeabilization conditions (0.1-0.5% Triton X-100)

  • Test antibody concentration range (typically starting with 1:100-1:500 dilution)

  • Optimize incubation time and temperature

• Multiplexing strategies:

  • Combine LIMK2 antibodies with phospho-cofilin antibodies to assess functional activity

  • Co-stain with phalloidin to visualize actin cytoskeleton

  • Include nuclear counterstains for automated cell segmentation

• Readout measurements:

  • Quantify subcellular localization (nuclear/cytoplasmic ratio)

  • Measure signal intensity as a proxy for expression level

  • Analyze co-localization with cytoskeletal components or other markers

• Quality control considerations:

  • Include positive and negative controls on each plate

  • Implement automated image quality assessment

  • Use machine learning algorithms for consistent feature extraction

This approach enables researchers to analyze LIMK2 expression, localization, and activity across large sample sets or in drug screening campaigns targeting cytoskeletal dynamics.

What emerging tools and techniques are enhancing LIMK2 research beyond traditional antibody applications?

Several cutting-edge technologies are expanding LIMK2 research capabilities:

• CRISPR-Cas9 gene editing:

  • Generation of LIMK2 knockout cell lines for functional studies

  • Knock-in of tagged LIMK2 variants for live-cell imaging

  • Creation of isoform-specific knockout models

• Proximity labeling techniques:

  • BioID or TurboID fusions with LIMK2 to identify proximal interaction partners

  • APEX2-LIMK2 fusions for capturing transient interactions

  • Spatial-specific interactome mapping in different subcellular compartments

• Advanced imaging approaches:

  • Super-resolution microscopy of LIMK2-cytoskeletal interactions

  • Live-cell FRET sensors for monitoring LIMK2 activity in real-time

  • Light-inducible LIMK2 activation systems

• Single-cell technologies:

  • Single-cell RNA-seq to examine LIMK2 expression heterogeneity

  • CyTOF with phospho-LIMK2 antibodies for multiparameter analysis

  • Digital spatial profiling for LIMK2 in tissue microenvironments

• Structural biology advances:

  • Cryo-EM studies of LIMK2 in complex with substrates

  • NMR analysis of LIM domain interactions

  • Structure-guided design of selective LIMK2 inhibitors

These emerging technologies complement traditional antibody applications and provide unprecedented insights into LIMK2 biology and therapeutic targeting.

How should researchers interpret contradictory findings regarding LIMK2's role across different cancer types?

When confronting seemingly contradictory findings about LIMK2 across cancer types, researchers should consider several contextual factors: