Phospho-CTNNB1 (S33/S37) Recombinant Monoclonal Antibody

What is the biological significance of CTNNB1 phosphorylation at S33/S37 sites?

CTNNB1 (beta-catenin) phosphorylation at S33/S37 residues represents a critical regulatory mechanism within the canonical Wnt signaling pathway. In the absence of Wnt ligands, CTNNB1 forms a complex with AXIN1, AXIN2, APC, CSNK1A1 (casein kinase 1α), and GSK3B (glycogen synthase kinase 3β) . Within this destruction complex, CSNK1A1 first phosphorylates CTNNB1 at serine 45 (S45), which serves as a priming event for subsequent sequential phosphorylation at T41, S37, and S33 by GSK3A/GSK3B . The phosphorylation at S33 and S37 specifically creates a recognition site for the SCF-β-TrCP ubiquitin ligase complex, which targets CTNNB1 for proteasomal degradation . This phosphorylation-dependent degradation mechanism prevents nuclear accumulation of CTNNB1 and thereby inhibits transcription of Wnt target genes. Consequently, mutations affecting these phosphorylation sites or dysregulation of this process are implicated in various cancers, including colon cancer, melanoma, and prostate cancer .

How do Phospho-CTNNB1 (S33/S37) antibodies differ from other beta-catenin antibodies?

Phospho-CTNNB1 (S33/S37) antibodies are specifically designed to recognize beta-catenin only when phosphorylated at serine residues 33 and 37, making them fundamentally different from pan-CTNNB1 antibodies that recognize total beta-catenin regardless of phosphorylation status . This specificity allows researchers to distinguish between the phosphorylated form destined for degradation and the non-phosphorylated, transcriptionally active form. Some antibodies may target only S33 or S37 individually, while others (like those described in the search results) recognize both phosphorylated residues simultaneously . Additionally, there are antibodies that recognize the extended phosphodegron including S33/S37/T41, which provides information about the complete GSK3-mediated phosphorylation pattern . The production method also differs between antibodies - monoclonal antibodies like the BC-22 clone offer high specificity and reproducibility, while polyclonal antibodies may provide broader epitope recognition but potentially with more batch-to-batch variation .

What are the primary applications of Phospho-CTNNB1 (S33/S37) antibodies in research?

Phospho-CTNNB1 (S33/S37) antibodies serve multiple critical applications in research investigating Wnt signaling dynamics. The primary applications include:

• Western blotting (WB): For quantifying phosphorylated CTNNB1 levels in cell or tissue lysates, especially when studying the effects of Wnt pathway activators or inhibitors .

• Immunofluorescence (IF): For visualizing the subcellular localization of phosphorylated CTNNB1, with recommended dilutions typically ranging from 1:20 to 1:200 .

• ELISA: For sensitive quantification of phosphorylated CTNNB1 in complex biological samples .

• Analyzing Wnt pathway activation: For distinguishing between active and inactive states of the pathway by measuring the ratio of phosphorylated to non-phosphorylated CTNNB1 .

• Studying disease mechanisms: For investigating aberrant Wnt signaling in various pathologies, including neurodevelopmental disorders (with spastic diplegia and visual defects) and cancers (particularly colon, prostate, and endometrial cancers) .

These antibodies are particularly valuable for monitoring CTNNB1 degradation dynamics in response to various experimental manipulations, including genetic knockouts of pathway components or pharmacological interventions .

How are recombinant monoclonal antibodies against Phospho-CTNNB1 (S33/S37) produced?

The production of recombinant monoclonal antibodies against Phospho-CTNNB1 (S33/S37) involves a sophisticated multi-step process combining immunological techniques with molecular biology methods. Initially, an animal is immunized with a synthesized peptide derived specifically from human Phospho-CTNNB1 (S33/S37) . Following immune response development, B cells (splenocytes) producing the desired antibody are isolated and their RNA extracted . This RNA undergoes reverse transcription to obtain cDNA encoding the antibody heavy and light chains. The antibody gene sequences are then identified through screening and sequencing procedures .

The selected heavy and light chain sequences are amplified via PCR and cloned into appropriate plasma expression vectors. These vector constructs are subsequently transfected into mammalian cells for recombinant protein production . The secreted antibody in the culture medium undergoes purification through affinity chromatography to obtain the final product - a highly specific recombinant monoclonal antibody against Phospho-CTNNB1 (S33/S37) . This recombinant production approach offers advantages over traditional hybridoma methods, including better reproducibility, elimination of animal maintenance for antibody production, and the potential for engineering modifications to enhance specificity or functionality.

How can Phospho-CTNNB1 (S33/S37) antibodies be used to study the kinetics of Wnt pathway activation?

The study of Wnt pathway activation kinetics using Phospho-CTNNB1 (S33/S37) antibodies requires a sophisticated experimental approach that capitalizes on the temporal dynamics of beta-catenin phosphorylation and degradation. To effectively investigate these kinetics, researchers should implement a time-course analysis following Wnt ligand stimulation or pathway inhibitor treatment, with samples collected at precisely timed intervals (typically 0, 15, 30, 60, 120, and 240 minutes) . This approach allows for the visualization of the rapid changes in phosphorylation status.

An alternative approach involves quantifying non-phospho-CTNNB1 (the species NOT phosphorylated at S33/S37/T41) as an inverse readout of phosphorylation status . This method has demonstrated higher sensitivity to condition-dependent changes compared to measuring total CTNNB1 alone. For rigorous mechanistic studies, combining these antibody-based approaches with small molecule inhibitors (such as CHIR-99021 for GSK3A/GSK3B inhibition) enables precise dissection of the phosphorylation cascade and its regulation .

What are the technical challenges in detecting phosphorylated CTNNB1 and how can they be overcome?

Detecting phosphorylated CTNNB1 presents several significant technical challenges that require careful methodological consideration. The primary challenges include:

• Rapid degradation: Phosphorylated CTNNB1 (at S33/S37) is quickly targeted for proteasomal degradation, resulting in extremely low steady-state levels that may fall below detection thresholds . To address this challenge, researchers can:

  • Employ proteasome inhibitors (e.g., MG132 or bortezomib) prior to cell lysis, though this approach requires careful control experiments as it may affect other pathway components .

  • Use highly sensitive detection methods such as enhanced chemiluminescence or fluorescently-tagged secondary antibodies.

  • Focus on measuring non-phosphorylated CTNNB1 as an inverse indicator of phosphorylation status .

• Phosphatase activity: Endogenous phosphatases in cell lysates can dephosphorylate CTNNB1 during sample preparation, leading to false-negative results. This can be mitigated by:

  • Including robust phosphatase inhibitor cocktails in all lysis buffers.

  • Maintaining samples at cold temperatures throughout processing.

  • Using denaturing lysis conditions when compatible with downstream applications.

• Cross-reactivity concerns: Some phospho-specific antibodies may recognize similar phosphorylation motifs on other proteins. Researchers should:

  • Validate antibody specificity using appropriate controls (phosphatase-treated samples, CTNNB1 knockdown cells).

  • Consider using multiple antibodies targeting different epitopes for confirmation.

  • Implement proper blocking procedures to minimize non-specific binding.

• Subcellular localization complexity: Phosphorylated CTNNB1 may be distributed across different cellular compartments with varying extraction efficiencies. This challenge can be addressed by:

  • Performing subcellular fractionation before analysis.

  • Using immunofluorescence techniques to visualize spatial distribution.

  • Employing specialized extraction buffers optimized for different cellular compartments.

How do mutations in the phosphodegron region affect antibody recognition and Wnt signaling outcomes?

Mutations in the phosphodegron region of CTNNB1, particularly those affecting residues S33, S37, T41, and S45, have profound implications for both antibody recognition and Wnt signaling outcomes. These mutations frequently occur in various cancers and can significantly alter pathway regulation . From an antibody recognition perspective, point mutations at S33 or S37 (the epitopes recognized by Phospho-CTNNB1 (S33/S37) antibodies) directly prevent phosphorylation at these sites and consequently abolish antibody binding . This creates a technically challenging scenario where the absence of signal could indicate either pathway activation (normal biology) or the presence of mutations (pathology).

The functional consequences of these mutations are equally significant. When S33 or S37 residues are mutated, GSK3B cannot phosphorylate these sites even when the priming phosphorylation at S45 by CSNK1A1 occurs normally . This prevents recognition by the SCF-β-TrCP ubiquitin ligase complex, resulting in stabilization of CTNNB1 and constitutive pathway activation independent of Wnt ligand stimulation . Importantly, different mutations within the phosphodegron can have varying effects on signal intensity. For example, mutations at S33 may allow partial phosphorylation at S37, resulting in reduced but not eliminated degradation.

To effectively study systems with potential phosphodegron mutations, researchers should implement a comprehensive analysis approach that includes:

• Genotyping the CTNNB1 phosphodegron region by sequencing

• Using antibodies targeting multiple phosphorylation sites separately

• Comparing phospho-specific antibody signals with total CTNNB1 levels

• Employing functional readouts of Wnt pathway activation (e.g., TCF/LEF reporters)

• Utilizing non-phospho-CTNNB1 antibodies to detect stabilized forms resulting from mutations

What is the relationship between HUWE1 and CTNNB1 phosphorylation, and how can it be investigated using phospho-specific antibodies?

The relationship between HUWE1 (HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1) and CTNNB1 phosphorylation represents a sophisticated regulatory mechanism in Wnt signaling that can be effectively investigated using phospho-specific antibodies. Research indicates that HUWE1 enhances WNT signaling by influencing the phosphorylation status of CTNNB1 at the critical S33/S37/T41 residues .

In CSNK1A1 knockout cells, where the priming phosphorylation at S45 is absent, HUWE1 appears to inhibit the GSK3A/GSK3B-mediated phosphorylation of the S33/S37/T41 residues . Experimental evidence shows that HUWE1 loss in CSNK1A1 knockout cells reduces non-phospho-CTNNB1 abundance by approximately 37%, which correlates closely with a similar reduction in soluble CTNNB1 . This suggests that HUWE1 normally prevents excessive phosphorylation of CTNNB1 at these residues.

To investigate this relationship using phospho-specific antibodies, researchers can implement several methodological approaches:

• Comparative analysis using both phospho-specific (S33/S37) and non-phospho-CTNNB1 antibodies in wildtype, HUWE1 knockout, CSNK1A1 knockout, and double knockout cellular models .

• Complementation experiments with GSK3A/GSK3B inhibitors (such as CHIR-99021) to verify the mechanism. Treatment of CSNK1A1 knockout; HUWE1 knockout cells with CHIR-99021 increased non-phospho-CTNNB1 abundance by 3.2-fold, suggesting GSK3A/GSK3B indeed mediates the increased phosphorylation observed after HUWE1 loss .

• Functional validation using WNT reporter assays to correlate changes in phosphorylation status with transcriptional outcomes. GSK3A/GSK3B inhibition in CSNK1A1 knockout; HUWE1 knockout cells increased WNT reporter activity by 10.9-fold, restoring signaling to levels comparable to CSNK1A1 knockout cells with intact HUWE1 .

• Time-course analysis following proteasome inhibition to capture the dynamic relationship between HUWE1 activity and phosphorylation-dependent CTNNB1 degradation.

This experimental framework utilizing phospho-specific antibodies enables precise dissection of how HUWE1 interfaces with the canonical CTNNB1 phosphorylation cascade to modulate Wnt signaling.

What are the optimal sample preparation methods for detecting phosphorylated CTNNB1?

Detecting phosphorylated CTNNB1 requires meticulous sample preparation to preserve phosphorylation status and ensure reliable results. The following methodological workflow is recommended:

• Cell harvest and lysis considerations:

  • Perform rapid harvesting to minimize post-harvest signaling changes

  • Use ice-cold phosphate-buffered saline for all washing steps

  • Add phosphatase inhibitor cocktails (containing sodium fluoride, sodium orthovanadate, sodium pyrophosphate, and β-glycerophosphate) to all buffers

  • Include protease inhibitors to prevent degradation of CTNNB1

  • Consider flash-freezing samples in liquid nitrogen if immediate processing is not possible

• Lysis buffer composition:

  • RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) with phosphatase and protease inhibitors works well for most applications

  • For challenging samples, consider using specialized buffers containing 8M urea or 6M guanidine hydrochloride to ensure complete protein denaturation and phosphoepitope exposure

• Sample processing:

  • Maintain constant cold temperature (4°C) during all processing steps

  • Limit freeze-thaw cycles to prevent phosphoepitope degradation

  • Sonicate samples briefly to shear DNA and ensure complete lysis

  • Centrifuge at high speed (≥14,000 × g) to remove insoluble material

  • Determine protein concentration using methods compatible with phosphatase inhibitors (Bradford or BCA assay)

• Special considerations for specific applications:

  • For Western blotting: Include phosphorylated protein standards as positive controls

  • For immunoprecipitation: Use antibody-conjugated beads with gentle agitation overnight at 4°C

  • For immunofluorescence: Fix samples rapidly with paraformaldehyde (avoid methanol fixation which can disrupt phosphoepitopes)

• Alternative approaches for challenging scenarios:

  • If direct detection of phosphorylated CTNNB1 is difficult due to rapid degradation, quantify non-phospho-CTNNB1 as an inverse measure

  • Consider phosphatase treatment of control samples to validate antibody specificity

  • When comparing conditions, standardize harvest timing carefully as phosphorylation states can change rapidly

How can researchers validate the specificity of Phospho-CTNNB1 (S33/S37) antibodies?

Validating the specificity of Phospho-CTNNB1 (S33/S37) antibodies is essential for ensuring experimental rigor and data reliability. Researchers should implement a multi-faceted validation strategy:

• Phosphatase treatment controls:

  • Divide samples into treated and untreated groups

  • Treat one set with lambda phosphatase to remove phosphate groups

  • The phospho-specific antibody signal should disappear in treated samples while total CTNNB1 signal remains unchanged

  • This confirms that the antibody specifically recognizes the phosphorylated form

• Genetic validation approaches:

  • Use CTNNB1 knockout or knockdown cells as negative controls

  • Generate point mutations at S33 and S37 (S33A/S37A) to create non-phosphorylatable CTNNB1

  • Compare antibody reactivity between wild-type and mutant proteins

  • In valid antibodies, signal should be absent in the mutant constructs

• Pharmacological manipulation:

  • Treat cells with GSK3 inhibitors like CHIR-99021 to reduce S33/S37 phosphorylation

  • Treat with Wnt pathway activators to reduce phosphorylation

  • Treat with Wnt pathway inhibitors to increase phosphorylation

  • A specific antibody will show corresponding signal changes aligned with the expected biology

• Peptide competition assays:

  • Pre-incubate the antibody with excess phosphorylated peptide containing the S33/S37 epitope

  • Pre-incubate with non-phosphorylated peptide as control

  • The phospho-peptide should block specific binding while the non-phospho-peptide should not

  • This confirms epitope-specific recognition

• Cross-validation with multiple antibodies:

  • Compare results using different phospho-CTNNB1 antibodies from diverse suppliers

  • Use antibodies with different epitope recognition (e.g., monoclonal vs polyclonal)

  • Consistent results across different antibodies increase confidence in specificity

• Validation across applications:

  • Test antibody performance in multiple techniques (WB, IF, IP, ELISA)

  • Confirm that results are consistent across methodologies

  • Document optimal working conditions for each application

What experimental controls are critical when studying CTNNB1 phosphorylation in the Wnt signaling pathway?

Studying CTNNB1 phosphorylation in the Wnt signaling pathway requires a comprehensive set of experimental controls to ensure data validity and interpretability. Critical controls include:

• Pathway manipulation controls:

  • Positive control: WNT ligand stimulation (e.g., WNT3A) to reduce CTNNB1 phosphorylation

  • Negative control: WNT pathway inhibitors to increase phosphorylation

  • Dose-response relationships should be established for all pathway modulators

  • Time-course experiments to capture dynamic changes in phosphorylation status

• Kinase and phosphatase controls:

  • GSK3 inhibition (e.g., CHIR-99021) to block phosphorylation at S33/S37/T41

  • CSNK1A1 inhibition to prevent priming phosphorylation at S45

  • Phosphatase inhibitors to preserve phosphorylation during sample preparation

  • Phosphatase treatment of control samples to validate antibody specificity

• Genetic controls:

  • CTNNB1 knockout/knockdown cells as negative controls for antibody specificity

  • CSNK1A1 knockout cells to study priming-independent phosphorylation

  • GSK3A/GSK3B knockout or knockdown to confirm kinase specificity

  • Rescue experiments with wild-type vs. phospho-mutant CTNNB1

• Detection controls:

  • Measurement of total CTNNB1 alongside phosphorylated forms

  • Quantification of non-phospho-CTNNB1 as a complementary readout

  • Parallel assessment of downstream transcriptional targets via reporter assays

  • Subcellular fractionation to distinguish cytoplasmic from nuclear CTNNB1 pools

• Technical controls:

  • Loading controls (e.g., GAPDH, β-actin) to normalize protein amounts

  • Recombinant phosphorylated and non-phosphorylated CTNNB1 as standards

  • Secondary antibody-only controls to assess non-specific binding

  • Multiple biological and technical replicates to ensure reproducibility

• Contextual controls:

  • Cell density standardization (Wnt signaling can be affected by cell-cell contacts)

  • Serum starvation before treatment to reduce baseline pathway activity

  • Vehicle controls for all treatments

  • Cell type comparisons to assess context-dependency of observations

How can researchers effectively interpret contradictory results in CTNNB1 phosphorylation studies?

Interpreting contradictory results in CTNNB1 phosphorylation studies requires systematic analysis of potential methodological and biological factors that may influence experimental outcomes. When faced with conflicting data, researchers should employ the following analytical framework:

• Methodological discrepancy analysis:

  • Antibody factors: Different antibodies may recognize distinct epitopes or have varying specificities. Compare antibody clones, host species, and manufacturing methods .

  • Detection sensitivity variations: Western blotting, immunofluorescence, and ELISA have different detection thresholds. Quantify lower limits of detection for each method used .

  • Sample preparation differences: Variations in lysis buffers, phosphatase inhibitors, and protein extraction protocols can significantly impact phosphoepitope preservation .

  • Timing considerations: CTNNB1 phosphorylation is highly dynamic. Standardize time points for cell harvesting after treatments.

• Biological context assessment:

  • Cell line differences: Various cell types may have distinct baseline Wnt pathway activity or CTNNB1 regulation mechanisms.

  • Genetic background variations: Mutations in Wnt pathway components may affect CTNNB1 phosphorylation dynamics .

  • Microenvironment factors: Cell density, extracellular matrix, and paracrine signaling can influence Wnt pathway activity.

  • Temporal dynamics: Contradictory results may reflect different points in a complex temporal response rather than true contradictions .

• Integrative analytical approaches:

  • Multi-antibody strategy: Use multiple phospho-specific antibodies targeting different epitopes or from different suppliers.

  • Complementary readouts: Combine direct phospho-CTNNB1 detection with measurements of non-phospho-CTNNB1 and total CTNNB1 .

  • Functional correlation: Assess whether phosphorylation changes correlate with expected functional outcomes (e.g., transcriptional reporter activity) .

  • Quantitative analysis: Apply statistical methods to determine if apparent contradictions are statistically significant or within normal experimental variation.

• Resolution strategies for specific contradiction types:

  Contradiction Type	Probable Causes	Resolution Strategy
  Phospho-signal present despite pathway activation	Incomplete pathway activation; Subpopulation effects	Single-cell analysis; Subcellular fractionation
  No phospho-signal despite pathway inhibition	Rapid degradation; Antibody sensitivity issues	Proteasome inhibition; Alternative detection methods
  Discrepancy between phospho-status and functional readouts	Parallel pathways; Post-translational modifications	Pathway component knockdowns; Mass spectrometry analysis
  Inconsistent results between replicates	Technical variability; Unstable reagents	Standardized protocols; Fresh reagent preparation

• Validation in multiple experimental systems:

  • Cross-validate findings in different cell lines

  • Confirm in vitro observations in appropriate in vivo models when possible

  • Use both gain-of-function and loss-of-function approaches

How can Phospho-CTNNB1 (S33/S37) antibodies be used in cancer research and diagnostics?

Phospho-CTNNB1 (S33/S37) antibodies offer powerful tools for cancer research and potentially diagnostics, given the critical role of aberrant Wnt signaling in tumorigenesis. In research applications, these antibodies enable:

• Identification of dysregulated Wnt signaling: By comparing phospho-CTNNB1 (S33/S37) levels between normal and cancer tissues, researchers can identify tumors with altered Wnt pathway activity. Reduced phosphorylation suggests pathway hyperactivation, which occurs in many cancer types including colon, prostate, and endometrial cancers .

• Mutation screening: CTNNB1 mutations affecting the phosphodegron region (particularly S33/S37) are common in certain cancers. Phospho-specific antibodies can serve as an immunohistochemical surrogate to identify samples likely harboring these mutations before confirmation by sequencing . The absence of phospho-signal in the presence of high total CTNNB1 suggests mutations in the phosphorylation sites.

• Therapeutic response monitoring: As pharmacological Wnt pathway inhibitors enter clinical trials, phospho-CTNNB1 antibodies provide valuable biomarkers for assessing target engagement and efficacy. Restoration of CTNNB1 phosphorylation following treatment would indicate successful pathway inhibition.

• Stratification of cancer subtypes: Different patterns of CTNNB1 phosphorylation may correlate with specific cancer subtypes or prognosis. For example, research suggests that nuclear β-catenin expression (which inversely correlates with phosphorylation) may be considered in risk stratification for endometrial cancer .

• Mechanism-of-action studies: When investigating novel compounds affecting the Wnt pathway, phospho-CTNNB1 antibodies help determine whether the mechanism involves regulation of the destruction complex or operates through alternative means.

For potential diagnostic applications, standardized protocols using phospho-CTNNB1 antibodies could assist in:

• Distinguishing between Wnt-driven and Wnt-independent tumors to guide treatment selection

• Identifying patients likely to respond to emerging Wnt pathway inhibitors

• Monitoring for acquired resistance mechanisms during treatment

• Complementing genetic testing for CTNNB1 mutations with functional protein assessment

Future development of multiplexed immunoassays combining phospho-CTNNB1 with other Wnt pathway components could enhance diagnostic precision and therapeutic decision-making in oncology.

What emerging techniques might enhance the utility of Phospho-CTNNB1 (S33/S37) antibodies in research?

Several emerging techniques hold promise for significantly enhancing the utility of Phospho-CTNNB1 (S33/S37) antibodies in research by improving sensitivity, specificity, spatial resolution, and throughput:

• Proximity ligation assays (PLA): This technique can detect phosphorylated CTNNB1 with significantly higher sensitivity than conventional immunoassays by generating an amplifiable DNA signal only when two antibodies bind in close proximity. Implementing PLA with antibodies targeting total CTNNB1 and phospho-S33/S37 would enable detection of low-abundance phosphorylated species and visualization of their precise subcellular localization.

• Mass cytometry (CyTOF): By combining phospho-CTNNB1 antibodies with metal-isotope labeling, researchers can simultaneously measure multiple phosphorylation sites and pathway components at the single-cell level. This approach would reveal heterogeneity in CTNNB1 phosphorylation within cell populations and correlate these patterns with other signaling events.

• Super-resolution microscopy: Techniques such as STORM, PALM, or STED microscopy paired with phospho-CTNNB1 antibodies could resolve the spatial organization of destruction complexes and phosphorylated CTNNB1 at nanometer resolution, providing unprecedented insights into the physical context of CTNNB1 phosphorylation events.

• Intracellular phospho-protein reporters: Development of genetically encoded biosensors that undergo conformational changes upon binding by phospho-CTNNB1 antibody fragments would enable real-time monitoring of phosphorylation dynamics in living cells.

• Automated high-content imaging platforms: Integration of phospho-CTNNB1 antibodies into high-throughput imaging systems would facilitate large-scale screens for compounds or genetic factors affecting CTNNB1 phosphorylation patterns.

• Phospho-proteomic integration: Combining targeted phospho-CTNNB1 antibody-based assays with global phospho-proteomic profiling would contextualize CTNNB1 phosphorylation within broader signaling networks.

• Spatial transcriptomics correlation: Coupling phospho-CTNNB1 immunostaining with spatial transcriptomics techniques would allow researchers to directly link phosphorylation status to Wnt target gene expression patterns at the tissue level.

• Nanobody-based detection systems: Development of nanobodies (single-domain antibody fragments) against phospho-CTNNB1 epitopes would enable superior tissue penetration, reduced background, and potentially intracellular expression for live-cell imaging.

These advanced methodologies, when optimized for phospho-CTNNB1 detection, promise to provide deeper mechanistic insights into the spatial, temporal, and contextual aspects of CTNNB1 phosphorylation in both normal and pathological settings.