CKS1B Antibody

What is CKS1B and what is its biological function?

CKS1B, a member of the conserved cyclin kinase subunit 1 (CKS1) protein family, functions as an essential regulatory component in cell cycle progression. It binds to the catalytic subunit of cyclin-dependent kinases and is essential for their biological function . The most well-characterized role of CKS1B is facilitating the ubiquitination and proteasomal degradation of p27Kip1, a key cell cycle inhibitor . This 10 kDa protein plays a critical role in regulating cell proliferation through its effects on cell cycle checkpoints . Research has demonstrated that CKS1B overexpression contributes to increased p27Kip1 turnover, enhanced cell proliferation, and poor prognosis in multiple cancer types .

What applications are validated for CKS1B antibodies?

CKS1B antibodies have been validated for multiple experimental applications, with specific recommendations for optimal results:

It is essential to optimize the antibody concentration for each specific testing system. Dilution requirements may be sample-dependent, and researchers should reference validation data from manufacturers for specific cell lines or tissue types .

What cell lines have been validated for CKS1B antibody research?

Multiple human cell lines have demonstrated positive results with CKS1B antibodies:

These validated cell lines provide reliable positive controls for researchers establishing CKS1B detection protocols in their laboratory .

How does CKS1B expression correlate with p27Kip1 levels in cancer?

Research has established an important inverse correlation between CKS1B and p27Kip1 protein levels in cancer cells. In studies of multiple myeloma, Western blot analysis of nuclear extracts from plasma cells revealed:

• Strong correlation between CKS1B mRNA and protein levels

• No correlation between CDKN1B mRNA and p27Kip1 protein levels

• Inverse correlation between CKS1B and p27Kip1 protein expression

What are effective methods for studying CKS1B function in cancer progression?

Several experimental approaches have proven valuable for investigating CKS1B's roles in cancer:

• RNA Interference (RNAi):

  • shRNA-mediated silencing of CKS1B has been successfully employed in multiple myeloma cell lines including JJN3, OCI-MY5, and XG-1

  • Comparative studies with SKP2 silencing help distinguish CKS1B-specific functions

• Protein Stability Assays:

  • Cyclohexamide treatment to measure p27Kip1 half-life following CKS1B manipulation

• Cell Proliferation and Viability Assays:

  • Trypan blue exclusion assays reveal that CKS1B silencing typically shows effects on cell viability after day 3 post-infection

  • By day 5, significant decreases in cell viability (44-49% vs 86-93% in controls) can be observed

• Apoptosis Assessment:

  • Western blotting for cleaved caspase-3 and PARP

  • Caspase inhibition assays using pan-caspase inhibitors like Z-VAD-fmk (100 μM)

• Expression of Modified p27Kip1:

  • Transfection with non-degradable p27Kip1 (p27 T187A) to bypass CKS1B-mediated degradation

These methodological approaches have revealed that CKS1B functions extend beyond simply regulating p27Kip1 degradation, with roles in apoptosis regulation and other cellular processes.

How does CKS1B influence the tumor immune microenvironment?

Recent research has uncovered a significant relationship between CKS1B and the tumor immune microenvironment, particularly in pancreatic cancer :

• CKS1B and PD-L1 Connection:

  • CKS1B knockdown suppresses PD-L1 levels in pancreatic cancer cells

  • This suggests CKS1B may contribute to immune evasion by promoting PD-L1 expression

• Signaling Pathway Involvement:

  • CKS1B appears to influence PD-L1 expression through the STAT3 signaling pathway

  • This is evidenced by changes in phosphorylated STAT3 levels following CKS1B manipulation

• Autophagy Regulation:

  • CKS1B knockdown affects LC3B expression, suggesting a role in regulating autophagy

These findings indicate that CKS1B, traditionally viewed as a cell cycle regulator, plays a more complex role in tumor biology by influencing both cancer cell properties and the immune microenvironment . This opens potential therapeutic avenues combining CKS1B targeting with immunotherapy approaches.

What is the recommended Western blotting protocol for CKS1B antibodies?

A standardized Western blotting protocol for CKS1B detection includes these critical steps:

• Sample Preparation:

  • For compartment-specific analysis, isolate cytosolic and nuclear fractions using appropriate fractionation kits

  • Perform protein quantification using BCA or similar assays

• Gel Electrophoresis:

  • Separate equal amounts of protein lysates on 4-12% SDS-PAGE gels

  • Given CKS1B's small size (10 kDa), ensure appropriate gel percentage and running conditions

• Transfer and Blocking:

  • Transfer proteins to PVDF membrane

  • Block membranes using 5% non-fat milk

• Primary Antibody Incubation:

  • Dilute CKS1B antibody at 1:1000-1:4000 (product-dependent)

  • Incubate membranes overnight at 4°C

• Detection:

  • Use appropriate HRP-conjugated secondary antibodies

  • Develop using ECL and a chemiluminescence detection system

• Expected Results:

  • The predicted band size for CKS1B is approximately 10 kDa

  • Some antibodies may show observed bands at slightly different sizes (e.g., <19 kDa)

  • Include appropriate controls such as Jurkat cell extracts

Always include loading controls (β-tubulin, GAPDH) and consider using the immunizing peptide as a negative control to confirm specificity .

How should CKS1B antibodies be stored and handled?

Proper storage and handling are crucial for maintaining antibody activity:

When working with CKS1B antibodies:

• Avoid repeated freeze-thaw cycles

• Briefly centrifuge the vial before opening

• When diluting, prepare only the amount needed for immediate experiments

• Follow safety precautions for antibodies containing sodium azide

What are effective approaches for immunohistochemical detection of CKS1B?

For tissue-based CKS1B analysis, immunohistochemistry provides spatial information about protein expression:

• Sample Preparation:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues

  • Tissue microarrays for high-throughput analysis

• Protocol Overview:

  • Dewaxing in xylene and rehydrating in alcohol series

  • Blocking endogenous peroxidase activity

  • Primary antibody incubation (e.g., DF3221, Affinity Biosciences at 1:500 dilution) overnight at 4°C

  • HRP-conjugated secondary antibody incubation (37°C, 40 min)

  • DAB visualization (5 min)

  • Hematoxylin counterstaining (30 sec)

• Scoring Systems:

  • Evaluate staining intensity and percentage of positive cells

  • Consider digital pathology quantification for objective measurements

This approach has been successfully employed in pancreatic cancer tissues to evaluate CKS1B expression and correlate it with clinical parameters .

How can CKS1B be targeted therapeutically?

CKS1B has emerged as a promising therapeutic target, particularly in cancers where it is overexpressed:

• Direct Inhibition Strategies:

  • Small molecule inhibitors targeting CKS1B-SKP2 interaction

  • Approaches that stabilize p27Kip1 by preventing its ubiquitination

• Genetic Targeting Approaches:

  • shRNA delivery systems for CKS1B silencing has shown efficacy in multiple myeloma models

  • This approach demonstrated significant effects on cell viability and induction of apoptosis

• Combination Therapy Approaches:

  • CKS1B inhibition + proteasome inhibitors (e.g., bortezomib)

  • CKS1B inhibition + immune checkpoint inhibitors, given its influence on PD-L1 expression

• Assessment Methods:

  • p27Kip1 stabilization as a pharmacodynamic marker

  • Cell cycle analysis to confirm G1 arrest

  • Apoptosis measurement through caspase activation

Research indicates that targeting CKS1B may be particularly effective in multiple myeloma and pancreatic cancer, where CKS1B overexpression is associated with aggressive disease and poor prognosis .

What is the role of CKS1B in drug resistance?

CKS1B has been identified as a drug resistance-inducing gene in multiple cancer types:

• Resistance Mechanisms:

  • Promotion of cell survival pathways

  • Regulation of apoptotic machinery

  • Influence on DNA damage response

• Clinical Correlations:

  • CKS1B overexpression is associated with poor treatment outcomes

  • Higher expression correlates with aggressive disease phenotypes

• Targeting Strategies:

  • CKS1B inhibition may restore sensitivity to standard therapies

  • Combination approaches targeting both CKS1B and specific resistance pathways

Understanding CKS1B's role in treatment resistance provides opportunities for developing more effective therapeutic strategies for aggressive cancers .

What are emerging areas of CKS1B research?

Several promising research directions are emerging in the CKS1B field:

• Tumor Microenvironment Interactions:

  • Further characterization of CKS1B's influence on immune checkpoint molecules

  • Investigation of stromal interactions in the tumor microenvironment

• Biomarker Development:

  • Validation of CKS1B as a prognostic and predictive biomarker

  • Development of companion diagnostics for CKS1B-targeted therapies

• Novel Targeting Approaches:

  • Structure-based drug design targeting CKS1B-protein interactions

  • Targeted degradation approaches (e.g., PROTACs)

• Combination Strategies:

  • Rational combinations with immunotherapy given the PD-L1 connection

  • Integration with standard chemotherapy regimens

These research directions hold promise for translating basic CKS1B biology into clinical applications for cancer treatment.