LATS2 Antibody

What is LATS2 and why is it important in research?

LATS2 (Large Tumor Suppressor kinase 2) is a serine/threonine-protein kinase with a molecular mass of approximately 120-121 kDa that functions as a crucial component of the Hippo signaling pathway. It acts as a negative regulator of YAP1, playing a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis . LATS2 is particularly important in research due to its multiple functions in cellular processes including maintenance of genomic stability, cell cycle regulation, and tumor suppression. Recent studies have also revealed its role in innate immunity and metabolic rewiring in certain cancer subtypes , making it a significant target for both basic and translational research.

What types of LATS2 antibodies are commonly used in research?

Researchers typically use two main types of LATS2 antibodies:

• Polyclonal antibodies: Generated by immunizing animals (typically rabbits) with synthetic peptides corresponding to specific regions of human LATS2. These recognize multiple epitopes and are useful for applications like IHC-P and ICC/IF .

• Monoclonal antibodies: Produced from single B-cell clones, offering higher specificity and consistency between batches. Recombinant monoclonal antibodies, such as clone EPR23126-488, have been validated for IP and Western blot applications .

Additionally, researchers can select phospho-specific antibodies that detect specific phosphorylated residues of LATS2 (e.g., phospho-T1041), which are critical for monitoring LATS2 activation status in the Hippo pathway .

What are the common applications for LATS2 antibodies in research?

LATS2 antibodies are employed in multiple research applications:

• Western blotting: For detecting total LATS2 protein (~140 kDa) and assessing expression levels

• Immunoprecipitation: For studying protein-protein interactions involving LATS2

• Immunohistochemistry: For examining LATS2 expression in tissue samples

• Immunofluorescence: For visualizing subcellular localization (both nuclear and cytoplasmic)

• Flow cytometry: For analyzing LATS2 expression at the single-cell level

The choice of application should determine antibody selection, as not all antibodies perform equally across different techniques.

How can LATS2 antibodies be used to study the Hippo signaling pathway?

LATS2 antibodies are instrumental in dissecting Hippo pathway dynamics through multiple approaches:

• Phosphorylation status monitoring: Phospho-specific antibodies detecting LATS2 T1041 phosphorylation serve as direct readouts of LATS2 activation . This activation is typically initiated by upstream kinases MST1/2, allowing researchers to monitor pathway activity.

• Negative feedback investigation: YAP activation induces LATS2 expression, forming a negative feedback loop. Researchers can use LATS2 antibodies alongside YAP/TEAD antibodies to study this regulatory mechanism . For example, studies have shown that YAP activation dramatically increases LATS2 expression, with the TEAD-binding motif being critical for this effect .

• Downstream pathway analysis: Analyzing YAP phosphorylation following LATS2 manipulation provides insights into pathway integrity. LATS2 antibodies can be combined with phospho-YAP antibodies to create a complete picture of Hippo signaling dynamics in different cellular contexts.

What role does LATS2 play in cancer research and how are antibodies used to study this?

LATS2 has multiple tumor suppressive functions that researchers investigate using specialized antibody-based techniques:

• Expression correlation studies: LATS2 antibodies enable researchers to evaluate LATS2 expression in tumor versus normal tissue. Studies show significant down-regulation of LATS2 in human breast cancer, accelerating mammary tumorigenesis .

• Functional domain analysis: Different antibodies targeting specific domains help dissect kinase-dependent versus kinase-independent functions. Some tumor suppressive functions of LATS2 are independent of its enzyme activity, requiring domain-specific antibodies for comprehensive analysis .

• Pathway crosstalk investigation: LATS2 antibodies reveal interactions with other cancer-related pathways:

  • LATS2-ASK1-JNK axis: LATS2 promotes ASK1-mediated stress response in a kinase-independent manner

  • LATS2-PQBP1-cGAS pathway: LATS2 enhances innate immune responses to viral infection

  • LATS2-PPARγ connection: LATS2 depletion reduces PPARγ signaling in luminal B breast tumors

How can phospho-specific LATS2 antibodies elucidate activation mechanisms?

Phospho-specific antibodies targeting LATS2 provide crucial insights into activation mechanisms:

• Activation sequence mapping: By using antibodies against different phosphorylation sites (particularly T1041/T1079), researchers can determine the sequential activation steps in the Hippo pathway .

• Alternative activation detection: Not all LATS2 activation occurs through canonical MST1/2. Phospho-specific antibodies help identify non-canonical activation, such as through cell-contact inhibition or mechanical stimuli.

• Quantitative activation assessment: Using phospho-LATS2 to total LATS2 ratio provides a quantitative measure of pathway activity. This approach reveals that continuous YAP activation dramatically increases both LATS2 expression and its phosphorylated forms .

What are the critical factors for successful LATS2 antibody validation?

Thorough validation of LATS2 antibodies requires multiple complementary approaches:

• Knockout/knockdown controls: Testing antibodies in LATS2 knockout or knockdown cells is the gold standard for specificity validation. Several studies have established LATS2-KO THP1 and HEK293A cell lines for this purpose .

• Peptide competition assays: Preincubation with the immunizing peptide should abolish specific signals. This validation approach has been documented with antibodies like ab111054, where treatment with synthesized peptide eliminates specific staining in both immunofluorescence and IHC applications .

• Cross-reactivity assessment: Testing against related proteins (particularly LATS1) is essential due to their high homology. Some antibodies may detect both paralogs despite sequence differences.

• Multi-application validation: An antibody performing well in one application may fail in others, necessitating application-specific validation.

• Species cross-reactivity: Confirming reactivity across species when studying LATS2 in animal models is critical for translational research.

What are the optimal protocols for detecting LATS2 in different sample types?

Optimal detection protocols vary based on sample type and application:

For tissue samples (IHC-P):

• Fixation: 10% neutral buffered formalin fixation (12-24 hours)

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0)

• Blocking: 5% normal serum from the same species as the secondary antibody

• Primary antibody dilution: 1:50-1:100 for most LATS2 antibodies

• Detection: Use of high-sensitivity detection systems due to variable LATS2 expression levels

• Controls: Include normal skeletal muscle tissue as positive control

For cell-based assays (IF/ICC):

• Fixation: 4% paraformaldehyde (10-15 minutes)

• Permeabilization: 0.1-0.5% Triton X-100

• Blocking: 1-5% BSA in PBS

• Antibody dilution: Typically 1:100 for most LATS2 antibodies

• Nuclear counterstain: DAPI for visualizing nuclear localization of LATS2

For protein detection (Western blotting):

• Lysis buffer: RIPA buffer supplemented with phosphatase inhibitors

• Sample preparation: Heat samples at 95°C for 5 minutes in reducing conditions

• Running conditions: 7.5% or 4-12% gradient gels due to LATS2's high molecular weight

• Transfer: Wet transfer recommended due to large protein size

• Antibody dilution: 1:1000 is standard for most LATS2 antibodies

• Detection: ECL or fluorescence-based detection systems

How can researchers optimize co-immunoprecipitation protocols for studying LATS2 interactions?

Co-immunoprecipitation (co-IP) is crucial for studying LATS2 protein-protein interactions:

• Lysis buffer optimization:

  • For nuclear interactions (e.g., LATS2-SNAI1): Use nuclear extraction buffer with 0.1% NP-40

  • For cytoplasmic interactions: CHAPS-containing buffers (0.3%) preserve weaker interactions

  • Always include phosphatase inhibitors to maintain phosphorylation status

• Cross-linking considerations:

  • For transient interactions: Consider reversible cross-linking with DSP (dithiobis-succinimidyl propionate)

  • LATS2-ASK1 interactions benefit from mild cross-linking approaches

• Antibody selection:

  • Use antibodies validated specifically for IP applications

  • Typical dilution: 1:100 for IP applications

  • Recombinant monoclonal antibodies often provide more consistent IP results

• Validation controls:

  • IgG control is essential to identify non-specific binding

  • Input controls (5-10% of lysate) should be run alongside IP samples

  • Reciprocal IP (using antibodies against the interacting partner) confirms true interactions

• Detection methods:

  • For known interactions: Direct Western blot detection

  • For novel interactome studies: Mass spectrometry following IP

How can researchers troubleshoot weak or non-specific LATS2 antibody signals?

Common issues with LATS2 detection can be addressed through systematic troubleshooting:

For weak signals:

• Increase antibody concentration incrementally (e.g., from 1:1000 to 1:500)

• Extend primary antibody incubation time (overnight at 4°C)

• Enhance signal amplification using biotin-streptavidin systems

• Verify sample preparation – LATS2 is susceptible to degradation

• Check for low expression levels in your cell/tissue type; consider using positive control samples

For non-specific signals:

• Increase blocking stringency (5% BSA or 5% milk)

• Reduce primary antibody concentration

• Add 0.1% Tween-20 to washing buffers

• Include peptide competition controls to identify specific bands

• Use LATS2 knockout/knockdown samples as negative controls

For high background:

• Extend washing steps (at least 3×10 minutes)

• Filter antibody solutions before use

• Prepare fresh blocking solutions

• Use monoclonal antibodies if polyclonal antibodies show high background

What are common pitfalls when interpreting LATS2 antibody results in cancer studies?

Researchers should be aware of several interpretation challenges in cancer studies:

• Expression heterogeneity: LATS2 expression varies significantly within tumors. Single sample analysis may not represent the entire tumor. Consider multiple sampling and correlate with clinical parameters.

• Paralog confusion: LATS1 and LATS2 share high homology but have distinct functions in breast cancer . Ensure antibody specificity between these paralogs by using paralog-specific controls.

• Subcellular localization: LATS2 functions differently depending on nuclear versus cytoplasmic localization. Perform fractionation studies or detailed immunofluorescence analysis to determine localization patterns.

• Post-translational modifications: Phosphorylation status affects LATS2 function. Use phospho-specific antibodies alongside total LATS2 antibodies to obtain a complete picture .

• Context-dependent functions: LATS2 effects may differ between cancer types. In breast cancer, LATS2 depletion has distinct effects on luminal B tumors compared to other subtypes .

How should discrepancies between different LATS2 antibodies be reconciled?

When different antibodies yield conflicting results:

How are LATS2 antibodies being used to study innate immunity pathways?

Recent research has revealed unexpected roles for LATS2 in innate immunity:

• LATS2-PQBP1-cGAS axis in viral response: LATS2 interacts with PQBP1, enhancing cGAS-STING mediated innate immune responses to HIV-1 . Researchers use co-immunoprecipitation with LATS2 antibodies to detect this interaction, revealing that:

  • LATS2 can phosphorylate PQBP1

  • LATS2 kinase activity is essential for enhancing immune responses

  • LATS2 knockout reduces cGAMP synthesis during viral infection

• Monitoring ISG induction: LATS2 enhances the expression of interferon-stimulated genes like ISG54 and CXCL10 during viral infection . Researchers combine LATS2 antibodies with RT-PCR analysis of ISGs to establish this connection.

• Signaling pathway integration: LATS2 antibodies help map connections between the Hippo pathway and innate immune signaling networks, particularly at the level of TBK1 and IRF3 activation.

What insights do LATS2 antibodies provide about metabolic regulation in cancer?

LATS2 antibodies have revealed surprising connections to cancer metabolism:

• LATS2-PPARγ connection: In luminal B breast tumors, LATS2 depletion reduces PPARγ signaling . Researchers use LATS2 antibodies in combination with metabolic profiling to understand this relationship.

• Glycolytic rewiring detection: LATS2 depletion increases glycolysis in specific tumor types . Combined analysis using LATS2 antibodies and glycolytic enzyme detection helps establish this metabolic switch.

• Therapeutic vulnerability assessment: Pharmacological activation of PPARγ induces LATS2-dependent cell death in luminal B-derived cells . This finding emerged from studies using LATS2 antibodies to detect expression levels before and after treatment.

• Metabolic pathway crosstalk: LATS2 antibodies help researchers dissect interactions between the Hippo pathway and metabolic signaling networks, revealing novel therapeutic opportunities.

How can researchers leverage LATS2 antibodies to study its non-canonical functions?

Beyond the canonical Hippo pathway, LATS2 has several non-canonical functions that can be studied with specialized antibody approaches:

• LATS2-ASK1-JNK signaling: LATS2 promotes ASK1-mediated signaling in a kinase-independent manner . Researchers use:

  • Co-immunoprecipitation with LATS2 antibodies to detect ASK1 interaction

  • Phospho-specific antibodies against JNK to measure pathway activation

  • Kinase-dead LATS2 mutants to demonstrate kinase-independent functions

• DNA damage response roles: LATS2 prevents DNA damage-induced apoptosis at high cell density . Researchers use:

  • LATS2 antibodies to monitor expression changes following DNA damage

  • Co-IP to detect LATS2-c-Abl interactions

  • Phospho-specific antibodies to track c-Abl inhibition by LATS2

• Cell cycle regulation: LATS2 negatively regulates G1/S transition by down-regulating cyclin E/CDK2 . Researchers combine:

  • LATS2 antibodies with cell cycle markers

  • Time-course studies during cell cycle progression

  • Co-IP to detect cell cycle-specific protein complexes

How are LATS2 antibodies contributing to our understanding of tumor heterogeneity?

LATS2 antibodies are revealing important aspects of tumor heterogeneity:

• Paralog-specific functions: While both LATS1 and LATS2 are down-regulated in breast cancer, they influence tumor progression differently :

  • LATS2 depletion: Affects metabolic rewiring in luminal B tumors

  • LATS1 depletion: Augments cancer cell plasticity, skewing tumors toward basal-like features

  • These differences, detected using paralog-specific antibodies, explain differential therapeutic responses

• Context-dependent functions: The same LATS2 perturbation can have opposite effects in different cellular contexts. Multi-antibody approaches help resolve these apparent contradictions.

• Spatial heterogeneity: Multiplex immunofluorescence with LATS2 antibodies reveals spatial distribution patterns within tumors, correlating with regional phenotypic differences.

What are the latest methodological advances in LATS2 antibody-based detection?

Recent technological developments have enhanced LATS2 detection:

• Proximity ligation assays (PLA): These enable visualization of endogenous LATS2 protein interactions at individual interaction sites within cells. PLA has been used to validate LATS2-ASK1 interactions with high sensitivity .

• NanoLuc-based protein-fragment complementation: This technique detects PPIs between overexpressed proteins in live human cancer cells. NanoPCA has confirmed LATS2-ASK1 interactions with significant luminescence signals (FOC = 10.2 and 15.0 in HeLa and MCF7 cells, respectively) .

• Multiplexed immunofluorescence: Allows simultaneous detection of LATS2 alongside other Hippo pathway components or interacting partners in the same sample.

• Live-cell imaging: Emerging antibody-based biosensors enable real-time tracking of LATS2 activity in living cells.

• Single-cell approaches: Combining LATS2 antibodies with single-cell technologies provides insights into cellular heterogeneity within tissues or tumors.

How can researchers integrate LATS2 antibody data with other omics approaches?

Multi-omics integration enhances the value of LATS2 antibody-based research:

• Proteogenomic correlation: Correlating LATS2 protein levels (detected via antibodies) with genomic alterations and transcriptomic profiles provides a comprehensive view of regulation.

• Phosphoproteomics integration: Combining phospho-specific LATS2 antibody data with global phosphoproteomics reveals pathway-wide changes following LATS2 activation or inhibition.

• Metabolism-proteomics interface: Integrating LATS2 antibody-based protein quantification with metabolomics has revealed unexpected roles in metabolic regulation, particularly in breast cancer .

• Spatial transcriptomics correlation: Aligning LATS2 antibody-based spatial data with spatial transcriptomics provides insights into regional tumor biology.

• Network analysis approaches: Computational integration of LATS2 interactome data (derived from antibody-based IP-MS studies) with transcriptional networks identifies regulatory hubs and potential therapeutic targets.