KLK11 Antibody

What is KLK11 and what cellular pathways does it influence?

KLK11 is a kallikrein-like serine protease that exists in two alternatively spliced isoforms: brain-type and prostate-type. It belongs to the kallikrein family of proteases that perform various physiological functions. In terms of cellular pathways, KLK11 has been demonstrated to influence several signaling cascades depending on tissue context .

In cancer biology, KLK11 can suppress cell proliferation in esophageal squamous cell carcinoma (ESCC) by inhibiting the Wnt/β-catenin signaling pathway . This inhibition results in decreased expression of downstream targets like cyclin D1 and Ki67, ultimately reducing cellular proliferation. Immunohistochemistry analysis has confirmed significantly lower KLK11 expression in ESCC tumor tissues compared to adjacent non-tumor tissues, suggesting its tumor-suppressive role in this context .

Conversely, in cardiovascular tissue, KLK11 promotes cardiomyocyte hypertrophy by activating the AKT-mTOR signaling pathway, which stimulates protein synthesis through regulation of S6K1 and 4EBP1 machinery . This dual nature of KLK11's influence on different signaling pathways makes it a fascinating subject for pathway-focused research.

How can researchers distinguish between normal and pathological KLK11 expression?

Researchers seeking to differentiate between normal and pathological KLK11 expression should employ multiple complementary approaches. Quantitative analysis using RT-qPCR enables precise measurement of KLK11 mRNA levels, while Western blotting provides information about protein abundance . When comparing expression between samples, statistical significance should be determined using appropriate tests (p<0.01 is typically considered significant for KLK11 expression differences) .

For tissue-specific expression analysis, immunohistochemistry offers spatial information about KLK11 distribution. In ESCC research, for example, immunoreactivity scoring systems have been developed to classify KLK11 expression levels. Studies have shown that KLK11 expression is dysregulated in approximately 67% of ESCC samples, providing a benchmark for evaluating expression patterns .

For serum-based analysis, particularly in cancer screening applications, researchers should note that elevated KLK11 serum levels occur in approximately 70% of women with ovarian cancer and 60% of men with prostate cancer . These biomarker thresholds can serve as reference points when establishing detection parameters for distinguishing between normal and pathological expression.

What are the recommended validation methods for confirming KLK11 antibody specificity?

Validation of KLK11 antibody specificity requires a multi-faceted approach. Begin with positive and negative control tissues with known KLK11 expression profiles. Human prostate and ovarian cancer tissues are suitable positive controls, while matched adjacent non-tumor tissues can serve as comparative references .

Antibody specificity should be confirmed through both genetic and biochemical approaches. Genetically, researchers should establish knockout or knockdown systems using shRNA targeting KLK11. The search results demonstrate this approach was successful in cellular models, where KLK11 shRNA significantly reduced both mRNA and protein expression levels . Western blotting of these knockdown systems should show correspondingly reduced signal intensity when probed with the KLK11 antibody.

For biochemical validation, peptide competition assays can determine whether the antibody binding is specific to KLK11. Additionally, cross-reactivity with other kallikrein family members should be assessed, particularly given the protein's multiple aliases and isoforms (Hippostasin, hK11, kallikrein 11, etc.) . When reporting validation results, provide Uniprot IDs (Human: Q9UBX7, Mouse: Q9QYN3) and Entrez Gene IDs (Human: 11012, Mouse: 56538) to ensure precise identification of the target protein .

How should researchers design experiments to investigate KLK11's seemingly contradictory roles in different cancer types?

KLK11 exhibits context-dependent functions across cancer types, necessitating carefully designed comparative studies. To investigate these contradictory roles, researchers should implement parallel experiments across multiple cancer cell lines with baseline KLK11 expression characterization. The experimental design should include:

First, establish a panel of cell lines representing different cancer types where KLK11 has shown variable expression patterns. Based on the literature, this panel should include esophageal squamous cell carcinoma lines (where KLK11 is downregulated and acts as a tumor suppressor) and colorectal cancer lines (where KLK11 upregulation is associated with chemoresistance) .

Second, employ both gain-of-function and loss-of-function approaches within each cancer type. For gain-of-function studies, researchers have successfully restored KLK11 expression in cell lines using transfection techniques, as demonstrated in TE-1 and EC18 ESCC cells . For loss-of-function studies, lentivirus-based RNA interference has proven effective in achieving stable KLK11 knockdown, with validation by both RT-qPCR and Western blotting .

Third, examine pathway-specific effects using reporter assays. Since KLK11 affects the Wnt/β-catenin pathway in ESCC and the PI3K/AKT pathway in colorectal cancer, researchers should implement TCF/LEF reporters for Wnt activity and phospho-AKT detection for PI3K/AKT activation . Include pathway inhibitors (e.g., XAV-939 for Wnt/β-catenin) as controls to confirm pathway specificity .

Finally, validate findings in patient samples representing different cancer types, correlating KLK11 expression with clinical parameters including tumor stage, metastatic status, and patient outcomes.

What methodological approaches can resolve contradictory findings regarding KLK11's role in drug resistance?

To address contradictory findings regarding KLK11's role in drug resistance, researchers should implement a systematic approach combining multiple methods and controlled experimental conditions:

Begin by establishing drug-resistant cell lines through standardized protocols. The search results describe a validated method for developing oxaliplatin-resistant colorectal cancer cells (HCT-8/L-OHP) by exposing parental cells to incrementally increasing concentrations of the drug . The resistance index (RI) should be calculated to quantify the degree of resistance acquired.

Quantify KLK11 expression changes during resistance development using both mRNA (RT-qPCR) and protein (Western blot) detection methods. Research indicates KLK11 is significantly upregulated in oxaliplatin-resistant colorectal cancer cells compared to parental lines (p<0.01) . Parallel analysis of known resistance-associated genes (e.g., ABCC1, ABCB1, GSTP1, ERCC1) provides context for evaluating KLK11's specific contribution .

Employ targeted manipulation of KLK11 expression in resistant cells using multiple independent shRNA constructs to control for off-target effects. Researchers have successfully used this approach, identifying KLK11 KD2 as the most effective knockdown construct following validation . After KLK11 knockdown, measure changes in drug sensitivity using standardized MTT assays across a range of drug concentrations (e.g., 10, 20, 40, 80, and 160 μmol/L oxaliplatin) .

Assess mechanistic changes following KLK11 modulation through apoptosis detection (flow cytometry), pathway analysis (Western blotting for PI3K/AKT signaling components), and downstream target evaluation (Bcl-2/Bax ratio, caspase-3 activity) . These mechanistic insights help explain functional observations and resolve apparent contradictions in KLK11's role.

How can researchers optimize KLK11 antibody-based detection in heterogeneous tumor samples?

Optimizing KLK11 antibody-based detection in heterogeneous tumor samples requires addressing several technical challenges:

Implement antigen retrieval optimization for immunohistochemistry applications. Since KLK11 detection sensitivity varies across tissue types, compare multiple antigen retrieval methods (heat-induced epitope retrieval with citrate buffer, EDTA buffer, or enzymatic retrieval) to determine optimal conditions for each tissue type . Researchers have successfully used this approach to detect differential KLK11 expression between tumor and adjacent tissues in ESCC samples .

Account for splice variant detection by selecting antibodies recognizing conserved regions present in both brain-type and prostate-type isoforms of KLK11 . When analyzing tissues that might express both variants, consider complementing protein detection with isoform-specific PCR to determine which isoform predominates in your sample.

For low-abundance detection, implement signal amplification methods such as tyramide signal amplification for immunohistochemistry or highly sensitive chemiluminescent substrates for Western blotting. These approaches have enabled detection of reduced KLK11 expression in ESCC tumor tissues compared to adjacent non-tumor tissues .

Validate antibody performance through knockdown controls in relevant cell lines before tissue application. Researchers have demonstrated successful antibody validation using lentivirus-mediated KLK11 knockdown models, which provide essential negative controls for antibody specificity confirmation .

What experimental models are most appropriate for studying KLK11's role in cardiac hypertrophy?

Investigating KLK11's role in cardiac hypertrophy requires both in vivo and in vitro experimental models that recapitulate key pathophysiological features:

For in vivo studies, the transverse aortic constriction (TAC) mouse model has proven effective in demonstrating KLK11 upregulation in hypertrophic hearts . This model allows researchers to measure functional cardiac parameters (fraction shortening, ejection fraction) and correlate them with KLK11 expression levels. For genetic manipulation in vivo, adeno-associated virus 9 delivery systems have successfully knocked down KLK11 expression in mouse hearts, enabling evaluation of the protein's role in hypertrophy development .

In vitro cardiomyocyte models should employ angiotensin II stimulation to induce hypertrophy, as this approach has successfully demonstrated KLK11's role in cardiomyocyte size regulation and hypertrophy-related fetal gene expression . When establishing these models, researchers should quantify cardiomyocyte size changes and analyze the expression of established hypertrophy markers.

Comparative studies across human and animal samples strengthen translational relevance. The literature demonstrates that KLK11 mRNA and protein levels are upregulated in both human hypertrophic hearts and experimental mouse models, validating the cross-species significance of this protein in cardiac pathology .

For mechanistic studies focusing on protein synthesis regulation, incorporate S6K1 and 4EBP1 activity measurements, as these have been identified as key effectors through which KLK11 modulates cardiac hypertrophy .

How should researchers analyze the interaction between KLK11 and the AKT-mTOR pathway in cardiac cells?

Analyzing KLK11's interaction with the AKT-mTOR pathway in cardiac cells requires a systematic approach combining genetic manipulation, pharmacological intervention, and pathway component analysis:

Begin with genetic modulation of KLK11 through both overexpression and knockdown approaches in cardiomyocytes. Evidence indicates that KLK11 overexpression promotes whereas knockdown represses cardiomyocyte hypertrophy induced by angiotensin II . Following genetic manipulation, analyze phosphorylation status of key AKT-mTOR pathway components using phospho-specific antibodies against AKT, mTOR, S6K1, and 4EBP1.

Incorporate pathway inhibitors as experimental controls. mTOR inhibitors (e.g., rapamycin) and AKT inhibitors should be used to confirm pathway specificity and determine whether KLK11's effects are dependent on AKT-mTOR signaling. The observed effects of KLK11 on cardiomyocyte hypertrophy occur through regulation of S6K1 and 4EBP1, key components of the mTOR pathway .

Employ co-immunoprecipitation or proximity ligation assays to investigate potential direct interactions between KLK11 and components of the AKT-mTOR pathway, which may reveal whether KLK11's effects are mediated through direct physical interactions or indirect signaling mechanisms.

What are the key technical considerations when measuring KLK11-mediated effects on protein synthesis in cardiomyocytes?

Accurately measuring KLK11-mediated effects on protein synthesis in cardiomyocytes requires attention to several technical considerations:

Select appropriate protein synthesis detection methods based on experimental objectives. For global protein synthesis measurements, surface sensing of translation (SUnSET) using puromycin labeling offers a non-radioactive alternative to traditional [35S]-methionine incorporation. For pathway-specific analysis, phosphorylation status of S6K1 and 4EBP1 serves as reliable proxies for protein synthesis machinery activation .

Control for confounding factors affecting protein synthesis rates. Standardize serum starvation periods before stimulation, as basal protein synthesis rates are influenced by growth factors in serum. Additionally, normalize protein synthesis measurements to cell number or total protein content to account for differences in cell density or size.

Implement time-course analyses to capture both acute and chronic effects of KLK11 modulation. KLK11's effects on protein synthesis may vary temporally, with different mechanisms predominating at different time points after stimulation or genetic manipulation.

Include appropriate controls for distinguishing between protein synthesis and degradation effects. Proteasome inhibitors (e.g., MG132) or autophagy inhibitors (e.g., bafilomycin A1) can help determine whether observed changes in protein content result from altered synthesis or degradation rates. This distinction is crucial when interpreting KLK11's effects on cardiomyocyte hypertrophy .

For in vivo validation, combine echocardiographic measurements of cardiac function with molecular analyses of hypertrophy markers and protein synthesis machinery. This multi-parameter approach provides a comprehensive assessment of KLK11's effects on cardiac remodeling .

How can researchers reconcile conflicting data on KLK11 functioning as both tumor suppressor and oncogene?

The dual nature of KLK11 as both tumor suppressor and oncogene presents a complex research challenge requiring sophisticated experimental approaches to reconcile conflicting data:

Conduct comprehensive tissue-specific profiling across multiple cancer types. Evidence indicates KLK11 suppresses esophageal squamous cell carcinoma by inhibiting the Wnt/β-catenin pathway , while promoting chemoresistance in colorectal cancer through PI3K/AKT signaling . This tissue-specific functional divergence may explain apparent contradictions in the literature.

Implement pathway-specific reporter systems to elucidate context-dependent mechanisms. For Wnt/β-catenin signaling, TCF/LEF reporters can confirm KLK11's inhibitory effects in ESCC . For PI3K/AKT pathway activation in colorectal cancer, phospho-AKT detection provides mechanistic insights into KLK11's pro-survival effects . When conducting these experiments, include pathway inhibitors (e.g., XAV-939 for Wnt/β-catenin) as controls to verify specificity .

Design epistasis experiments that sequentially manipulate KLK11 and downstream effectors to establish causal relationships. For example, in ESCC models, KLK11 knockdown followed by β-catenin knockdown would test whether β-catenin is required for the effects of KLK11 deficiency . Such experiments help establish mechanistic hierarchies that explain contextual differences.

Utilize advanced genetic models incorporating inducible, tissue-specific expression systems. These allow precise temporal and spatial control of KLK11 expression, enabling dissection of primary versus compensatory effects that might contribute to seemingly contradictory observations across different experimental systems.

What experimental design is recommended for investigating KLK11 antibody cross-reactivity with other kallikrein family members?

Investigating potential cross-reactivity between KLK11 antibodies and other kallikrein family members requires a systematic approach to ensure specificity:

Begin with in silico analysis comparing sequence homology between KLK11 and other kallikrein family members, particularly focusing on the epitope region recognized by the antibody. KLK11 has multiple aliases (Hippostasin, hK11, kallikrein 11, etc.) , which may complicate literature comparison without proper identification using UniProt IDs (Human: Q9UBX7, Mouse: Q9QYN3) .

Implement a panel-based validation approach using cell lines with manipulated expression of KLK11 and closely related kallikreins. This panel should include: 1) Wild-type cells with normal kallikrein expression; 2) KLK11 knockdown cells created using validated shRNA constructs ; 3) Cells overexpressing KLK11; and 4) Cells overexpressing closely related kallikreins with structural similarity to KLK11.

Employ multiple antibody-based techniques with varying stringency. Western blotting under reducing and non-reducing conditions can reveal cross-reactivity that might be masked under standard conditions. Immunoprecipitation followed by mass spectrometry analysis provides definitive identification of all proteins recognized by the antibody.

Utilize peptide competition assays with both KLK11-specific peptides and peptides derived from related kallikreins. These assays can quantitatively determine relative binding affinities and reveal potential cross-reactivity. Decreasing signal intensity with increasing KLK11-specific peptide concentration confirms antibody specificity.

What troubleshooting approaches are recommended when KLK11 antibody yields inconsistent results across different experimental platforms?

When facing inconsistent results with KLK11 antibodies across different experimental platforms, implement this systematic troubleshooting approach:

Evaluate antibody compatibility with specific sample preparation methods. KLK11 detection sensitivity may vary depending on fixation methods (for immunohistochemistry) or lysis buffers (for Western blotting). Compare multiple preparation protocols, as research groups have successfully detected KLK11 in both fixed tissues and cell lysates .

Optimize antibody concentration through titration experiments. The optimal antibody dilution may differ substantially between applications (Western blotting, immunohistochemistry, flow cytometry). Begin with the manufacturer's recommended concentration and perform serial dilutions to identify application-specific optimal concentrations.

Address potential epitope masking issues caused by protein interactions or post-translational modifications. Protein-protein interactions can shield antibody binding sites, while phosphorylation or other modifications may alter epitope recognition. For Western blotting, compare reducing and non-reducing conditions. For immunohistochemistry or immunofluorescence, test multiple antigen retrieval methods.

Implement validation controls appropriate for each experimental platform. For immunohistochemistry, include known positive (prostate or ovarian cancer tissues) and negative control tissues . For Western blotting, include lysates from cells with confirmed KLK11 knockdown . For flow cytometry, use isotype controls and cells with manipulated KLK11 expression.

Consider antibody lot-to-lot variability by recording lot numbers and repeating critical experiments when receiving new antibody lots. When possible, validate new lots against previously verified lots to ensure consistent performance across experiments.