CSNK1E Antibody

What is CSNK1E and why is it significant in research?

CSNK1E (Casein Kinase 1 Epsilon) is a serine/threonine protein kinase belonging to the casein kinase I family. It plays significant roles in various cellular processes including cell growth, development, differentiation, DNA damage repair, and cell cycle progression . It's particularly important in regulating cell signaling pathways that control cell proliferation and survival . CSNK1E is also crucial in delaying negative feedback signals within the transcription-translation-based autoregulatory loop that forms the core of the circadian mechanism . The dysregulation of CSNK1E has been associated with various diseases including cancer, neurological disorders (particularly Parkinson's disease), and metabolic syndromes, making it an important research target .

What are the optimal conditions for using CSNK1E antibodies in Western blotting?

For optimal Western blot results with CSNK1E antibodies:

How can I effectively use CSNK1E antibodies for visualizing subcellular localization?

CSNK1E primarily localizes to the cytoplasm and nucleus, but its distribution can change under different cellular conditions, particularly in disease states . For effective immunofluorescence or immunohistochemistry:

• Fixation methods: Both paraformaldehyde (for IF) and formalin-fixed paraffin-embedded (for IHC-P) tissues are compatible with most CSNK1E antibodies .

• Antigen retrieval: For IHC applications with CSNK1E antibodies, TE buffer pH 9.0 is recommended for antigen retrieval, though citrate buffer pH 6.0 can also be used as an alternative .

• Co-localization studies: When studying CSNK1E's role in Parkinson's disease or other conditions, co-staining with mitochondrial markers can reveal pathological mislocalization. Research shows that while wild-type CHCHD2 colocalizes with mitochondria, the T61I mutant associates with cytosolic CSNK1E in aggresomes .

• Controls for specificity: Use siRNA knockdown of CSNK1E as a negative control. Research has validated antibody specificity by showing disappearance of CSNK1E signals upon siRNA treatment .

How is CSNK1E involved in the pathogenesis of Parkinson's disease, and what methodologies can detect this association?

CSNK1E plays a critical role in the pathogenesis of Parkinson's disease (PD), particularly in cases involving CHCHD2 mutations:

• Mechanism of involvement: In PD with the CHCHD2 T61I mutation, the mutant protein mislocalizes from mitochondria to the cytosol where it recruits CSNK1E/D. These kinases then phosphorylate neurofilament and α-Synuclein, forming cytosolic aggresomes characteristic of PD .

• Detection methods:

  • Proximity assays: Close proximity assays can demonstrate physical interaction between CSNK1E and CHCHD2 T61I (but not wild-type CHCHD2) .

  • Immunoprecipitation followed by LC-MS/MS: This approach identified CSNK1E as the kinase preferentially interacting with CHCHD2 T61I .

  • Phospho-specific antibodies: Detection of phosphorylated α-Synuclein (particularly at position 129) can reveal CSNK1E activity .

• Experimental models: Both Chchd2 T61I knock-in and transgenic mice display neurodegenerative phenotypes with aggresomes containing Chchd2 T61I, Csnk1e/d, phospho-α-Synuclein, and phospho-neurofilament in dopaminergic neurons . Similar aggresomes are observable in postmortem PD patient brains and dopaminergic neurons generated from patient-derived iPS cells .

• Therapeutic relevance: CSNK1E/D inhibitors (like PF-670462) can suppress the phosphorylation of neurofilament and α-Synuclein, demonstrating therapeutic potential. These inhibitors improved neurodegenerative phenotypes in Chchd2 T61I mutant mice .

What methodologies can be used to study CSNK1E's role in cancer cell growth and survival?

CSNK1E has been identified as overexpressed in cancer samples compared to normal tissue across various tissue types, suggesting its significance as a potential therapeutic target . Research methodologies to study its role include:

• RNA interference approach: Using shRNA targeting CSNK1E (shCSNK1E) in various cancer cell lines demonstrated tumor-specific growth inhibition. Multiple shRNA clones targeting different regions of CSNK1E mRNA should be used to confirm target specificity .

• Validation approaches:

  • Real-time quantitative PCR: To confirm knockdown efficiency of CSNK1E mRNA levels .

  • Cell cycle analysis by flow cytometry: shCSNK1E treatment results in G2/M phase arrest in cancer cells .

  • Apoptosis detection: Through observation of sub-G1 population and PARP1 cleavage assays .

• Kinase inhibition studies: Pharmacological inhibitors like IC261 that target the kinase activity of CK1ε can recapitulate the effects of shRNA, confirming that inhibition of kinase activity (rather than other protein functions) is crucial for the observed anti-cancer effects .

• Molecular mechanism studies:

  • Gene expression analysis: shCSNK1E decreases mRNA levels of cyclin B1 and cyclin A2 while slightly increasing cyclin D1 levels, explaining the G2/M arrest phenotype .

  • Western blotting for activation markers: Detection of cleaved PARP1 and activated caspase-3 confirms apoptotic cell death mechanism .

• Selectivity testing: Test effects in paired normal vs. transformed cell lines (e.g., BJ-TERT vs. BJ-TERT/LT/ST/RAS V12) to confirm cancer-cell selectivity of targeting CSNK1E .

How can I differentiate between CSNK1E and its closely related family member CSNK1D in my experiments?

Distinguishing between these highly homologous family members requires careful experimental design:

• Antibody selection: Choose antibodies specifically validated for CSNK1E with minimal cross-reactivity to CSNK1D. Look for antibodies targeting non-conserved regions, typically in the variable N- and C-terminal non-catalytic domains where these isoforms differ significantly .

• RNAi validation: When using RNA interference, design multiple shRNAs targeting regions unique to CSNK1E. Validate specificity by qPCR using primers that can distinguish between the two isoforms .

• Functional validation: CSNK1E and CSNK1D may share substrates and perform overlapping biological roles. In some experimental contexts, silencing both together provides more complete inhibition of the biological function than targeting either alone .

• Chemical inhibitors: While some inhibitors like PF-670462 target both CSNK1E and CSNK1D, the differential effects observed between shRNAs targeting CK1δ versus CK1ε suggest isoform-specific functions that can be exploited experimentally .

• Expression patterns: Consider tissue-specific expression differences between the isoforms when interpreting results.

What are the recommended controls for validating CSNK1E antibody specificity in various applications?

Proper controls are essential for ensuring antibody specificity:

• Positive controls:

  • Cell/tissue types: A431 cells and mouse brain tissue have been validated as positive Western blot controls .

  • For IHC: Human breast cancer tissue, heart, kidney, testis, and ovary tissues have been validated for positive detection .

• Negative controls:

  • Genetic knockdown/knockout: siRNA or shRNA targeting CSNK1E provides an excellent specificity control. Multiple studies have validated antibody specificity by demonstrating signal disappearance following CSNK1E knockdown .

  • Peptide competition: Pre-incubation of the antibody with the immunizing peptide should eliminate specific staining.

  • Isotype controls: Use matched isotype antibodies (rabbit or mouse IgG depending on the host species) to control for non-specific binding .

• Molecular weight verification: The observed molecular weight for CSNK1E should be between 46-52 kDa . Bands at significantly different molecular weights may represent non-specific binding or post-translationally modified forms.

• Cross-species validation: If the antibody is expected to work across species, confirm specific detection in each species individually using appropriate positive controls.

• Multiple antibody validation: Using different antibodies targeting distinct epitopes of CSNK1E can provide confirmation of specificity, particularly in novel applications or model systems.

How can CSNK1E antibodies be used to investigate circadian rhythm regulation in experimental models?

CSNK1E plays a significant role in regulating circadian rhythms by delaying negative feedback signals within the transcription-translation-based autoregulatory loop . Research approaches include:

• Temporal expression analysis: Use CSNK1E antibodies in Western blot or immunofluorescence to track protein expression and localization changes throughout the circadian cycle in different tissues.

• Phosphorylation state monitoring: CSNK1E phosphorylates various clock proteins. Use phospho-specific antibodies alongside CSNK1E antibodies to correlate kinase localization with substrate phosphorylation.

• Pharmacological manipulation: Combine CSNK1E/D inhibitors like PF-670462 (which affects circadian rhythms) with immunostaining to correlate molecular changes with behavioral or cellular rhythm alterations .

• Genetic models: Compare CSNK1E localization and activity in wild-type versus clock gene mutant models to establish pathway relationships.

• Co-immunoprecipitation: Use CSNK1E antibodies to pull down protein complexes and identify circadian-regulated interaction partners under different conditions or time points.

What are the latest methodological approaches for studying CSNK1E's involvement in α-Synuclein phosphorylation and aggregation?

Recent research has revealed CSNK1E's critical role in α-Synuclein phosphorylation, particularly relevant to Parkinson's disease pathogenesis:

• In vitro kinase assays: Recombinant auto-active CSNK1E (R178C mutant) phosphorylates α-Synuclein at position 129, while non-active wild-type CSNK1E does not. This assay can be used to screen potential inhibitors .

• Phosphorylation site mapping: α-Synuclein contains consensus phosphorylation sequences for casein kinase, and various phospho-specific antibodies can be used to detect CSNK1E-mediated phosphorylation events .

• Aggresome visualization: Combined immunofluorescence using antibodies against CSNK1E, phospho-α-Synuclein, and aggresome markers can reveal the formation and composition of protein aggregates in cellular and animal models .

• Patient-derived models: Dopaminergic neurons generated from patient-derived iPS cells harboring CHCHD2 mutations show aggresomes containing CSNK1E and phospho-α-Synuclein, providing a valuable model system for mechanistic studies and therapeutic testing .

• Therapeutic intervention assessment: CSNK1E antibodies can be used to monitor the efficacy of CSNK1E/D inhibitors in reducing α-Synuclein phosphorylation and aggresome formation in various experimental models, including cultured cells and mouse models of Parkinson's disease .

How can CSNK1E antibodies be utilized in studying potential therapeutic approaches for neurodegenerative diseases?

CSNK1E antibodies offer several methodological approaches for investigating therapeutic strategies:

• Target engagement studies: Use CSNK1E antibodies to confirm binding and cellular localization of novel CSNK1E inhibitors through competition assays or changes in protein complexes.

• Pharmacodynamic markers: Monitor phosphorylation status of CSNK1E substrates (α-Synuclein, neurofilament) using phospho-specific antibodies to assess inhibitor efficacy in vivo and in vitro .

• Therapeutic combination assessment: CSNK1E antibodies can help evaluate synergistic effects when combining CSNK1E inhibitors with other therapeutic agents targeting different aspects of neurodegenerative pathways.

• Biomarker development: Investigate CSNK1E and its phosphorylated substrates as potential biomarkers for disease progression or treatment response using antibody-based detection methods.

• Model system validation: Use CSNK1E antibodies to confirm the relevance of various model systems (cell lines, patient-derived neurons, animal models) by comparing CSNK1E expression, localization, and activity with human disease samples .

What methodological considerations are important when studying CSNK1E isoforms and splice variants?

The CSNK1 family consists of at least seven isoforms (α, β, γ1, γ2, γ3, δ, and ε) with additional splice variants . When studying specific CSNK1E variants:

• Antibody epitope mapping: Ensure that the antibody's epitope is present in all splice variants of interest. Review the immunogen sequence information to determine which isoforms or splice variants will be recognized .

• Isoform specificity: The casein kinase I family members share highly conserved kinase domains but differ significantly in the length and primary structure of their amino- and carboxy-terminal non-catalytic domains . Select antibodies targeting these distinctive regions for isoform specificity.

• Molecular weight discrimination: Different splice variants may have slightly different molecular weights. Use high-resolution gels to separate closely related variants in Western blot applications.

• RT-PCR validation: Complement antibody-based detection with RT-PCR using primers specific to different splice variants to confirm expression patterns.

• Functional redundancy consideration: When investigating CSNK1E function, consider potential compensatory effects from other isoforms, particularly CSNK1D which shares substrates and functions with CSNK1E in some contexts .