CDKL3 Antibody

What is CDKL3 and what are its basic functions in cellular processes?

CDKL3 (Cyclin-dependent kinase-like 3) is a serine/threonine protein kinase that belongs to the CMGC Ser/Thr protein kinase superfamily, specifically within the cyclin-dependent kinase-like (CDKL) kinase subfamily. It shares conserved α-helix structures on the kinase domain which serve as putative cyclin-binding sites, similar to CDKs . CDKL3 contains a potential nucleus localization sequence (NLS) that is conserved across different species, enabling it to primarily localize in the nucleus with smaller amounts present in the cytoplasm .

Functionally, CDKL3 plays critical roles in:

• Cell cycle regulation and progression

• Cell proliferation and growth

• Signal transduction pathways, particularly those involving Akt/PKB

• Autophagy regulation

• Immunomodulation within tumor microenvironments

Recent studies have demonstrated that CDKL3 directly promotes cell cycle progression in cancer through two parallel regulatory mechanisms. First, it couples with cyclin A2 to directly lead to retinoblastoma (Rb) phosphorylation, facilitating G0/G1 to S phase transition. Second, it prevents the ubiquitin-proteasomal degradation of cyclin-dependent kinase 4 (CDK4) through direct phosphorylation on T172, which sustains G1 phase progression .

How do I select the appropriate CDKL3 antibody for my experimental needs?

Selection of the appropriate CDKL3 antibody requires consideration of multiple technical parameters:

Key Selection Criteria:

• Reactivity: Determine which species your samples originate from. Available CDKL3 antibodies include those specific for human, mouse, and rat CDKL3 .

• Application compatibility: Different experimental techniques require antibodies validated for specific applications:

  • Western blot (WB)

  • Immunohistochemistry (IHC)

  • Enzyme-linked immunosorbent assay (ELISA)

  • Immunofluorescence (IF)

• Clonality:

  • Monoclonal antibodies (e.g., 4F1 clone): Offer high specificity for a single epitope

  • Polyclonal antibodies: Recognize multiple epitopes, potentially providing stronger signals

• Host species: Consider the host species (mouse, rabbit) to avoid cross-reactivity issues in your experimental system .

Methodological Approach:

• Review the antibody's validation data, including images from western blots or IHC staining

• Check if the antibody recognizes the specific domain or region of CDKL3 relevant to your research

• Confirm the antibody works in your specific sample preparation conditions

• Consider performing parallel validation using multiple antibodies when establishing a new experimental system

What are the optimal conditions for CDKL3 antibody storage and handling?

For maximum stability and performance of CDKL3 antibodies, follow these evidence-based protocols:

Storage Conditions:

• Ship at 4°C

• Upon delivery, aliquot to minimize freeze-thaw cycles

• Store at -20°C for long-term preservation

• Avoid repeated freeze-thaw cycles as they can degrade antibody performance

Buffer Formulation:
The optimal buffer formulation for CDKL3 antibodies typically includes:

• Phosphate Buffered Saline (without Mg²⁺ and Ca²⁺)

• pH maintained at 7.4

• 150mM NaCl

• 0.02% Sodium Azide as preservative

• 50% Glycerol for stability

Working Dilutions:
Based on validated protocols, recommended working dilutions vary by application:

• IHC: 1:50-1:100

• ELISA: 1:5000

• Western blot: Typically 1:1000 (may vary by manufacturer)

To maintain antibody integrity, work in a clean environment, handle with care, and follow the manufacturer's specific recommendations for each antibody product.

How can I optimize Western blot protocols for CDKL3 detection?

Western blot optimization for CDKL3 detection requires attention to several parameters:

Protocol Optimization:

• Sample Preparation:

  • Extract total proteins using standard lysis buffers

  • Perform protein quantification using BCA protein assay kit

  • Load 20-40μg of protein per lane for optimal detection

• Gel Separation and Transfer:

  • Use 10% SDS-PAGE for optimal separation

  • Transfer to PVDF membranes (preferred over nitrocellulose for CDKL3)

  • Confirm transfer efficiency with reversible protein staining

• Blocking and Antibody Incubation:

  • Block membranes with 5% skim milk for 1 hour at room temperature

  • Incubate with primary CDKL3 antibody at 1:1000 dilution overnight at 4°C

  • Use appropriate HRP-coupled secondary antibody (typically 1:2000-1:5000)

• Detection Controls:

  • Include GAPDH (1:2000) as loading control

  • Consider positive control samples with known CDKL3 expression

  • Include molecular weight markers to confirm the expected size of 67kDa

Troubleshooting Common Issues:

• If signal is weak: Increase antibody concentration or extend incubation time

• If background is high: Increase washing steps or adjust blocking conditions

• If multiple bands appear: Validate with alternative antibodies or knockout controls

What are the best methods for validating CDKL3 antibody specificity?

Rigorous validation of CDKL3 antibody specificity is crucial for reliable experimental results:

Comprehensive Validation Strategy:

• Genetic Approaches:

  • CDKL3 knockdown or knockout: Compare antibody signal in wild-type versus CDKL3-depleted samples. Research shows that lentivirus-based CDKL3 knockdown significantly reduces detection signal in multiple cell lines .

  • Overexpression validation: Analyze signal in cells overexpressing tagged CDKL3 constructs

• Mass Spectrometry Validation:

  • Immunoprecipitate CDKL3 using the antibody

  • Analyze precipitated proteins by mass spectrometry to confirm CDKL3 identity

• Cross-Reactivity Assessment:

  • Test antibody against recombinant CDKL family members (CDKL1, CDKL2, CDKL4, CDKL5)

  • Evaluate performance in tissues/cells known to lack CDKL3 expression

• Multiple Antibody Concordance:

  • Compare signal patterns using different CDKL3 antibodies targeting distinct epitopes

  • Consistent pattern across antibodies increases confidence in specificity

• Immunohistochemical Controls:

  • Include peptide competition assays where the antibody is pre-incubated with the immunizing peptide

  • Include serial dilutions to confirm signal proportionality with antibody concentration

A comprehensive validation approach combining multiple methods provides the highest confidence in antibody specificity for CDKL3 detection.

How can I effectively use CDKL3 antibodies for immunofluorescence staining?

Effective immunofluorescence staining with CDKL3 antibodies requires precise optimization:

Immunofluorescence Protocol:

• Sample Preparation:

  • For cultured cells: Fix with 4% paraformaldehyde for 15 minutes

  • For tissue sections: Use 5-7μm sections mounted on positively charged slides

• Permeabilization and Blocking:

  • Permeabilize with 0.1% Triton X-100 for 10 minutes

  • Block with 5% normal serum (from the same species as secondary antibody) for 1 hour

• Antibody Incubation:

  • Apply primary CDKL3 antibody (1:100 dilution) overnight at 4°C

  • Wash extensively with PBS (3-5 times, 5 minutes each)

  • Apply fluorophore-conjugated secondary antibody (1:500) for 1 hour at room temperature

• Counterstaining and Mounting:

  • Counterstain nuclei with DAPI (1:1000 dilution)

  • Mount with anti-fade mounting medium to preserve fluorescence

  • Seal with nail polish to prevent drying

Expected Subcellular Localization Pattern:
Research has demonstrated that CDKL3 localizes predominantly in the nucleus with smaller amounts in the cytoplasm. This nuclear localization appears to be largely independent of cell cycle phase . Validation experiments should confirm this expected localization pattern.

Co-localization Studies:
Consider dual immunofluorescence staining with markers of:

• Nuclear structures (to confirm nuclear localization)

• Cell cycle phase markers (to assess cell cycle dependency)

• Potential interacting partners (e.g., Akt, Rb, CDK4)

How does CDKL3 contribute to tumor progression and what experimental approaches best demonstrate this?

CDKL3 has been implicated in promoting tumor progression through multiple mechanisms across several cancer types:

Key Mechanisms of CDKL3-Mediated Tumor Progression:

• Cell Cycle Regulation:

  • CDKL3 drives rapid cell cycle progression through direct phosphorylation of retinoblastoma (Rb) protein

  • It stabilizes CDK4 by preventing ubiquitin-proteasomal degradation through T172 phosphorylation

• Akt/PKB Pathway Activation:

  • In osteosarcoma, CDKL3 enhances Akt activation and its downstream effects

  • Co-immunoprecipitation experiments demonstrate that CDKL3 physically interacts with Akt1, specifically binding to its kinase domain

• Immunosuppressive Tumor Microenvironment Formation:

  • CDKL3 shapes an immunosuppressive tumor microenvironment by regulating autophagy

  • This contributes to reduced anti-tumor immunity and potentially decreased efficacy of immunotherapy

• Targeting Specific Oncogenic Pathways:

  • In glioma, CDKL3 regulates RRM2 and activates the JNK signaling pathway

  • Direct interaction between CDKL3 and RRM2 was confirmed through co-immunoprecipitation

Recommended Experimental Approaches:

These combined approaches provide comprehensive evidence for CDKL3's role in tumor progression and identify potential therapeutic targeting strategies.

What is the relationship between CDKL3 expression and autophagy in cancer, and how can this be investigated?

The relationship between CDKL3 and autophagy represents an emerging area of cancer research:

CDKL3-Autophagy Relationship in Cancer:

• Regulatory Mechanism:

  • CDKL3 regulates autophagy induction in cancer cells

  • CDKL3 expression correlates with autophagy-related gene expression patterns

  • Knockdown of CDKL3 affects autophagy flux and autophagosome formation

• Immunomodulatory Effects:

  • CDKL3-regulated autophagy influences macrophage polarization toward an immunosuppressive phenotype

  • ESCC cell supernatant after autophagy activation inhibits M1-type macrophage polarization and pro-inflammatory cytokine secretion

• Clinical Significance:

  • A CDKL3-related autophagy (CrA) risk score model has prognostic value in esophageal cancer

  • The CrA risk score correlates with M2 macrophage infiltration

Experimental Investigation Methods:

• Autophagy Marker Analysis:

  • Western blot analysis of LC3B conversion (LC3B-I to LC3B-II)

  • Immunofluorescence staining for LC3B puncta formation

  • Co-localization studies with other autophagy markers (p62/SQSTM1, ATG proteins)

• Autophagy Flux Assays:

  • Treatment with autophagy inhibitors (e.g., chloroquine, bafilomycin A1) to assess LC3B-II accumulation

  • Tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) to distinguish autophagosomes from autolysosomes

  • Long-lived protein degradation assays to measure autophagy-dependent protein turnover

• Molecular Interaction Studies:

  • Analysis of CDKL3 interaction with autophagy regulatory proteins

  • Identification of CDKL3 phosphorylation targets within the autophagy machinery

  • Development of phospho-specific antibodies to track CDKL3-mediated modifications

• Risk Model Construction:
  The CrA risk score model can be calculated using the following equation:

  CrA risk score = (0.380889116MAP1LC3B exp) + (-0.321484102TSC2 exp) + (0.846337455PPP2CA exp) + (-0.351584102UBE2J2 exp) + (-0.277601266ATM exp) + (0.354855247PIK3CB exp) + (0.450974523CTSD exp) + (-0.315185584ITPR3 exp) + (-0.327763971*ATG16L1 exp)

• Macrophage Polarization Assessment:

  • Flow cytometry analysis of macrophage markers (M1: CD86; M2: CD206)

  • qRT-PCR analysis of polarization markers and cytokines (M1: IL-12, TNF-α; M2: IL-10, TGF-β)

  • Co-culture experiments with CDKL3-modulated cancer cells and macrophages

This integrated approach enables comprehensive characterization of the complex relationship between CDKL3 expression and autophagy regulation in cancer contexts.

How can CDKL3 be targeted in cancer therapy and what methods are available to assess potential inhibitors?

Targeting CDKL3 represents a promising therapeutic strategy based on its established role in tumor progression:

Therapeutic Potential of CDKL3 Targeting:

• Rationale for Targeting:

  • Overexpression of CDKL3 correlates with poor prognosis in multiple cancers

  • CDKL3 drives proliferation through cell cycle regulation

  • CDKL3 contributes to immunosuppressive tumor microenvironment

  • CDKL3 activation of oncogenic pathways (Akt, JNK) promotes cancer progression

• Potential Therapeutic Approaches:

  • Small-molecule kinase inhibitors targeting CDKL3 catalytic activity

  • Degrader-based approaches (PROTACs) to reduce CDKL3 protein levels

  • siRNA/shRNA-based gene silencing strategies

  • Disruption of specific protein-protein interactions (e.g., CDKL3-cyclin A2, CDKL3-Akt1)

Methods to Assess CDKL3 Inhibitors:

• Combination Therapy Potential:

  • With immune checkpoint inhibitors: CDKL3 inhibition may enhance response to immunotherapy

  • With cell cycle inhibitors (CDK4/6 inhibitors): Potential synergistic effects

  • With Akt pathway inhibitors: Enhanced pathway suppression

  • With autophagy modulators: Exploitation of CDKL3-autophagy regulatory axis

• Biomarker Development:

  • CDKL3 expression levels as predictive biomarkers for therapy response

  • CDKL3-related autophagy (CrA) risk score for patient stratification

  • Integration with immunotherapy response biomarkers (TIDE, IPS scores)

Development of CDKL3-targeted therapies represents a promising area for cancer treatment, particularly for tumors with high CDKL3 expression and associated poor prognosis.

How does CDKL3 interact with the cell cycle machinery and what techniques are optimal for studying these interactions?

CDKL3 plays a crucial role in cell cycle regulation through specific molecular interactions:

CDKL3 Cell Cycle Interactions:

• Direct Interaction with Cyclins:

  • CDKL3 contains a conserved α-helix structure serving as a putative cyclin-binding site

  • CDKL3 couples with cyclin A2 to form a functional complex

  • This complex directly leads to retinoblastoma (Rb) phosphorylation, facilitating G0/G1 to S phase transition

• CDK4 Protein Stabilization:

  • CDKL3 directly phosphorylates CDK4 on threonine 172 (T172)

  • This phosphorylation prevents ubiquitin-proteasomal degradation of CDK4

  • Consequently, CDK4 protein levels are sustained, promoting G1 phase progression

• Nuclear Localization and Cell Cycle Independence:

  • CDKL3 contains a nuclear localization sequence (NLS) conserved across species

  • It primarily localizes in the nucleus with smaller amounts in the cytoplasm

  • This nuclear localization appears largely independent of cell cycle phase

Optimal Techniques for Studying CDKL3-Cell Cycle Interactions:

These techniques provide complementary approaches to dissect the molecular mechanisms by which CDKL3 regulates cell cycle progression, offering insights into potential therapeutic targeting strategies.

What is the significance of CDKL3 in Akt/PKB signaling and how can this interaction be experimentally validated?

CDKL3 plays a critical role in regulating the Akt/PKB signaling pathway, with significant implications for cancer progression:

CDKL3-Akt/PKB Signaling Relationship:

Experimental Validation Methods:

• Protein-Protein Interaction Analysis:

  • Co-immunoprecipitation (Co-IP) to detect CDKL3-Akt1 complexes

  • Domain mapping using truncated Akt1 constructs to identify interaction regions

  • Pull-down assays with recombinant proteins to confirm direct interaction

• Phosphorylation Studies:

  • Western blot analysis of Akt phosphorylation sites (Ser473, Thr308)

  • Phospho-specific antibodies to track activation status

  • Kinase assays to assess Akt activity in presence/absence of CDKL3

• Pharmacological Approaches:

  • Use of Akt inhibitors (e.g., MK2206) to determine if CDKL3's effects are Akt-dependent

  • PI3K inhibitors to assess upstream pathway involvement

  • mTOR inhibitors to examine downstream consequences

• Cellular Phenotype Rescue Experiments:

  • CDKL3 knockdown followed by constitutively active Akt expression

  • Akt knockdown followed by CDKL3 overexpression

  • Assessment of proliferation, migration, and survival phenotypes

• Immunohistochemical Analysis in Clinical Samples:

  • Correlation of CDKL3 expression with phospho-Akt levels

  • Multivariate analysis with survival outcomes

  • Tissue microarray analysis of multiple tumor types

The CDKL3-Akt interaction represents a therapeutically targetable vulnerability in cancers with CDKL3 overexpression, offering opportunities for the development of novel treatment strategies.

How does CDKL3 influence the tumor immune microenvironment and what experimental models best demonstrate this?

CDKL3 has emerged as a significant regulator of the tumor immune microenvironment, particularly through its effects on immune cell infiltration and function:

CDKL3's Influence on Tumor Immune Microenvironment:

• Immune Cell Infiltration:

  • CDKL3 expression correlates with tumor-infiltrating immune cell (TIIC) composition

  • High CDKL3 expression is associated with increased infiltration of immunosuppressive cells (M2 macrophages, regulatory T cells)

  • CDKL3 expression negatively correlates with cytotoxic immune cells (CD8+ T cells, NK cells)

• Immunosuppressive Mechanisms:

  • CDKL3 regulates autophagy to promote formation of immunosuppressive tumor microenvironment

  • CDKL3 expression positively correlates with inhibitory immune checkpoints

  • CDKL3 may be associated with hyperprogression in immunotherapy

• Immune Pathway Modulation:

  • Interferon (IFN) pathway is significantly activated upon CDKL3 knockdown

  • CDKL3 influences chemokine and chemokine receptor expression

  • High CDKL3 expression may attenuate anti-tumor immunity by inhibiting the IFN pathway

Optimal Experimental Models and Approaches:

Research Findings on CDKL3 and Immunotherapy Response:

Analysis of CDKL3 expression and immunotherapy response shows:

• Patients with low CDKL3 expression have significantly decreased TIDE scores and increased IPS scores

• TIDE prediction indicates patients with lower CDKL3 expression respond better to immunotherapy in multiple cohorts:

  • TCGA cohort: 57.0% (low CDKL3) vs. 42.4% (high CDKL3)

  • GSE53625 cohort: 53.3% (low CDKL3) vs. 22.5% (high CDKL3)

  • GSE165252 cohort: 40% (low CDKL3) vs. 21.4% (high CDKL3)

These findings suggest CDKL3 expression could serve as a potential biomarker for immunotherapy response prediction and targeting CDKL3 might enhance immunotherapy efficacy in cancer patients.

How can CDKL3 expression be used as a prognostic biomarker in cancer, and what validation steps are necessary?

CDKL3 has emerged as a promising prognostic biomarker across multiple cancer types:

CDKL3 as a Prognostic Biomarker:

Necessary Validation Steps:

Standardized IHC Scoring System:
For translational applications, a standardized CDKL3 IHC scoring system has been developed:

• Negative (-): No detectable CDKL3 staining

• Weak (+): Faint staining in <50% of cells

• Moderate (++): Moderate staining in >50% of cells or strong staining in <50% of cells

• Strong (+++): Strong staining in >50% of cells

This scoring system has shown correlation with clinical outcomes and can be implemented in pathology laboratories with appropriate training and quality control.

What are the potential advantages and limitations of targeting CDKL3 in cancer therapy?

Targeting CDKL3 in cancer therapy presents both promising opportunities and challenges:

Potential Advantages:

Limitations and Challenges:

• Target Validation Status:

  • While CDKL3 shows promise, it remains less validated compared to established cancer targets

  • Additional mechanistic studies and in vivo validation are needed

  • Comprehensive target validation across multiple cancer types is lacking

• Druggability Considerations:

  • The three-dimensional structure of CDKL3 has not been fully characterized

  • Selectivity against other CDKL family members and related kinases may be challenging

  • Development of highly specific inhibitors requires substantial medicinal chemistry efforts

• Potential Side Effects:

  • CDKL3's physiological roles in normal tissues remain incompletely understood

  • CDKL family members are involved in neurological functions (e.g., CDKL5 in neurological disorders)

  • Off-target effects on other CDKL family members might lead to unexpected toxicities

• Resistance Mechanisms:

  • Potential compensatory upregulation of related kinases

  • Bypass mechanisms through parallel pathways (e.g., other cell cycle regulators)

  • Acquired mutations in CDKL3 that prevent drug binding

Development Strategy Recommendations:

• Prioritize comprehensive structural studies to facilitate rational drug design

• Develop selective tool compounds to further validate CDKL3 as a therapeutic target

• Identify robust biomarkers for patient selection in clinical trials

• Consider combination approaches, particularly with immunotherapy

• Explore alternative targeting modalities (e.g., PROTACs, allosteric inhibitors)

The development of CDKL3-targeted therapies represents a promising but challenging opportunity in cancer therapeutics, requiring careful validation and strategic development approaches.

How can CDKL3 antibodies be effectively used in high-throughput screening platforms for drug discovery?

CDKL3 antibodies can serve as valuable tools in high-throughput screening (HTS) platforms for drug discovery:

Applications in Drug Discovery Screening:

• Target-Based Screening Approaches:

  • Kinase Activity Assays: Using phospho-specific antibodies to detect changes in CDKL3 substrates (e.g., CDK4-T172, Rb)

  • Binding Assays: Competitive binding assays to identify compounds displacing known CDKL3 interactors

  • Cellular Thermal Shift Assays (CETSA): Detection of compound-induced thermal stabilization of CDKL3 using specific antibodies

• Phenotypic Screening Applications:

  • High-Content Imaging: Using fluorescently-labeled CDKL3 antibodies to track subcellular localization changes upon compound treatment

  • Pathway Reporter Assays: Detection of downstream pathway modulation (Akt, cell cycle) in response to CDKL3 inhibition

  • Multiplex Profiling: Simultaneous detection of multiple pathway components using antibody arrays

• Target Validation Screening:

  • RNA Interference Screens: Correlation of CDKL3 knockdown phenotypes with compound effects

  • CRISPR-Based Screens: Identification of synthetic lethal interactions with CDKL3 inhibition

  • Resistance Mechanism Studies: Identification of bypass pathways using antibody-based detection methods

Methodological Considerations for HTS Implementation:

Optimization Strategies for Antibody-Based HTS:

• Antibody Selection Criteria:

  • Validate multiple antibodies to identify those with highest specificity and sensitivity

  • Select antibodies recognizing distinct epitopes for sandwich-based detection systems

  • Consider using monoclonal antibodies for higher reproducibility in large-scale screening

• Assay Development Considerations:

  • Optimize antibody concentration to maximize signal-to-noise ratio

  • Determine optimal incubation times and washing conditions

  • Establish robust positive and negative controls

  • Implement automated liquid handling to minimize variability

• Data Analysis Approaches:

  • Develop standardized quantification methods for antibody-based signals

  • Implement appropriate normalization strategies

  • Establish dose-response relationships for hit compounds

  • Incorporate machine learning algorithms for complex phenotypic analysis

• Validation of Screening Hits:

  • Confirm target engagement using orthogonal antibody-based methods

  • Validate hits across multiple cell lines with varying CDKL3 expression levels

  • Assess effects on known CDKL3 substrates and interacting partners

  • Perform selectivity profiling against related kinases