CYCB1-1 Antibody

What is the functional role of CYCB1-1 in cell cycle regulation?

CYCB1-1 is a B-type cyclin that forms active complexes with cyclin-dependent kinases (CDKs), particularly CDKB1 in plants. These complexes play crucial roles in cell cycle progression, specifically at the G2/M transition. Unlike the typical inhibition of CDKs upon DNA damage, CYCB1-1 is specifically activated after DNA damage to mediate homologous recombination (HR) repair . This represents a plant-specific mechanism where CDKB1-CYCB1 complexes are activated while the major mitotic cell-cycle activity is blocked after DNA damage, creating a division of labor strategy for DNA repair .

How does CYCB1-1 function differ between plants and mammals?

In plants, particularly Arabidopsis thaliana, CYCB1-1 forms active complexes with plant-specific B1-type CDKs (CDKB1s) to regulate homologous recombination . This complex can phosphorylate RAD51, a core mediator of HR . In mammals, Cyclin B1 primarily partners with CDK1 and controls mitotic entry. Importantly, Cyclin B1 is frequently overexpressed in several human cancers, including breast, prostate, and non-small cell lung cancers . While both plant and mammalian Cyclin B1 proteins function in cell cycle regulation, plants have evolved specific regulatory mechanisms involving SOG1 (a functional analog of p53) that directly controls CYCB1;1 expression after DNA damage .

How should I design experiments to study CYCB1-1 function in DNA damage response?

To effectively study CYCB1-1 function in DNA damage response, implement a multi-faceted approach:

• Genetic approach: Utilize cycb1 single and multiple mutants (such as cycb1;1, cycb1;3, and cycb1;1 cycb1;3 double mutants) alongside cdkb1 mutants to assess genetic interactions .

• DNA damage induction: Treat plants with DNA-damaging agents such as:

  • Cisplatin (15-50 μM) - forms DNA crosslinks

  • Bleomycin (0.6 μg/ml) - induces double-strand breaks

  • Hydroxyurea (1 mM) - inhibits DNA replication

• Phenotypic analysis: Measure root growth inhibition, which provides a quantitative readout of DNA damage sensitivity. Compare mutant lines to wild-type under control and treatment conditions .

• Cellular analysis: Perform propidium iodide staining to visualize cell death patterns in root tips .

• DNA damage quantification: Use gamma-H2AX immunostaining to quantify DNA damage foci, comparing wild-type and mutant responses during treatment and recovery periods .

What methods are most effective for visualizing CYCB1-1 expression and localization?

For optimal visualization of CYCB1-1 expression and localization, consider these methodological approaches:

• Reporter gene constructs: Generate transgenic lines containing the CYCB1;1 promoter (~1.2 kb 5′ region) fused to the N-terminal part of CYCB1;1 (including the destruction box) and GFP . This allows visualization of CYCB1;1 expression patterns and protein accumulation in living tissues.

• Immunofluorescence: Use CYCB1-1 antibodies for immunolocalization in fixed tissues. For optimal results:

  • Dilute antibodies 1:400 for immunofluorescence applications

  • Include appropriate negative controls (cycb1 mutants) and positive controls

• Co-localization studies: Perform double immunolabeling with markers of DNA damage (such as gamma-H2AX) or cell cycle phases to understand the spatial and temporal dynamics of CYCB1-1 in response to DNA damage .

• Live cell imaging: For dynamic studies, use time-lapse microscopy of GFP-tagged CYCB1-1 to monitor protein accumulation and degradation during the cell cycle and following DNA damage.

How can I design a homologous recombination assay to study CYCB1-1 function?

To assess CYCB1-1's role in homologous recombination, implement an in vivo HR assay system as follows:

• Reporter system: Utilize a disrupted beta-glucuronidase (uidA/GUS) gene as a substrate for homologous recombination. When HR occurs, the GUS gene is restored, allowing for histochemical detection as blue spots .

• Genetic background: Cross the HR reporter line with cycb1 mutants to generate plants homozygous for both the reporter and the respective mutation .

• DNA damage induction: Treat seedlings with increasing concentrations of DNA-damaging agents (e.g., 15 μM and 30 μM cisplatin for 3 days) .

• Quantification: Count the number of blue sectors per plant after histochemical staining to quantify HR events. Compare wild-type and mutant plants under both control and treatment conditions .

• Statistical analysis: Apply appropriate statistical tests (such as Student's t-test) to determine significant differences in HR frequencies between genotypes .

How do CDKB1-CYCB1 complexes regulate RAD51 during homologous recombination?

CDKB1-CYCB1 complexes directly regulate RAD51, a key mediator of homologous recombination, through the following mechanisms:

• Direct phosphorylation: CDKB1;1-CYCB1;1 complexes can phosphorylate RAD51 in vitro, suggesting a direct regulatory mechanism . The substrate preference of different CDK-cyclin complexes toward RAD51 varies, with CDKA;1-CYCB1;1 and CDKB1;1-CYCB1;1 showing the highest activity .

• RAD51 recruitment: In cdkb1;1 cdkb1;2 and cycb1;1 cycb1;3 double mutants, RAD51 foci formation is severely reduced after DNA damage, indicating that these complexes are required for proper RAD51 recruitment to damaged DNA . This effect is even more pronounced in cdkb1;1 cdkb1;2 double mutants, consistent with their severe sensitivity to cisplatin .

• Pathway specificity: The regulatory function appears to be specific to the CDKB1-CYCB1 pathway, as triple mutants (cycb1;1 cdkb1;1 cdkb1;2) show phenotypes similar to cdkb1;1 cdkb1;2 double mutants when exposed to DNA-damaging agents, suggesting they function in the same pathway .

What is the relationship between SOG1 and CYCB1-1 expression in DNA damage response?

The relationship between SOG1 (SUPPRESSOR OF GAMMA RESPONSE 1) and CYCB1-1 expression represents a critical regulatory mechanism in plant DNA damage response:

• Direct transcriptional regulation: SOG1, a plant-specific transcription factor that functions similarly to p53 in animals, directly controls CYCB1;1 expression after DNA damage . ChIP experiments with tagged SOG1 in plants grown on cisplatin demonstrate that SOG1 binds to the CYCB1;1 genomic region, particularly the 5′ and 3′ UTR .

• Specificity of regulation: Importantly, SOG1 specifically regulates CYCB1;1 but not other CYCB1 family members (CYCB1;2, CYCB1;3, CYCB1;4) . Similarly, SOG1 does not appear to bind to CDKB1;1 or CDKB1;2 genomic regions, suggesting these genes are regulated by different mechanisms .

• Checkpoint signaling: This regulatory relationship explains why CYCB1;1 upregulation after DNA damage depends on ATM and SOG1, establishing a direct link between DNA damage checkpoint signaling and the activation of HR repair machinery .

How do CYCB1 family members differ in their response to DNA damage?

CYCB1 family members show distinct patterns of regulation and function during DNA damage response:

• Differential expression: Only CYCB1;1 is significantly upregulated after cisplatin treatment, while CYCB1;2, CYCB1;3, and CYCB1;4 do not show increased expression based on reporter lines . This indicates a specialized function for CYCB1;1 in DNA damage response.

• Functional redundancy: Despite their different expression patterns, genetic analysis suggests some functional redundancy among CYCB1 family members. Single cycb1 mutants show relatively mild phenotypes on DNA-damaging agents, while cycb1;1 cycb1;3 double mutants exhibit enhanced sensitivity .

• Substrate specificity: When assembled into CDK complexes, CYCB1;1 shows higher activity toward RAD51 compared to other cyclins, suggesting specialized substrate recognition that may be unique to this family member .

How should I analyze and interpret gamma-H2AX foci data in CYCB1-1 studies?

Gamma-H2AX foci quantification is a powerful method to assess DNA damage levels, but requires careful analysis and interpretation:

• Quantification approach:

  • Count the number of distinct gamma-H2AX foci per nucleus

  • Analyze multiple cells (>100) across different experimental replicates

  • Apply statistical tests (e.g., Student's t-test) to determine significant differences between genotypes

• Interpretation guidelines:

  • Higher numbers of gamma-H2AX foci in cycb1 or cdkb1 mutants compared to wild-type after DNA damage treatment indicate defective DNA repair

  • Compare foci numbers during treatment and recovery periods to assess repair kinetics

  • Correlate foci numbers with other phenotypic data such as root growth inhibition or cell death

• Comparative analysis: When examining gamma-H2AX foci, include appropriate controls:

  • Wild-type plants (baseline response)

  • DNA repair mutants (e.g., atm, atr, rad51) as positive controls for increased DNA damage

  • Multiple time points to track the persistence of DNA damage

How do I resolve contradictory data regarding CYCB1-1 function?

When faced with contradictory results in CYCB1-1 functional studies, implement this systematic approach:

• Genetic background assessment: Ensure consistency in the genetic backgrounds used across experiments. Different ecotypes or the presence of unintended mutations can affect results.

• Treatment conditions comparison: Standardize experimental conditions:

  • DNA damage agent concentrations

  • Treatment duration

  • Plant age and growth conditions

  • Recovery periods after treatment

• Methodology validation: Apply multiple, complementary techniques to address the same question:

  • Combine genetic approaches with biochemical analysis

  • Use both reporter constructs and immunological detection

  • Verify antibody specificity using appropriate controls

• Quantitative analysis: Implement rigorous statistical analysis:

  • Ensure adequate sample sizes

  • Apply appropriate statistical tests

  • Consider biological and technical replicates separately

• Contextual interpretation: Consider that CYCB1-1 function may be context-dependent:

  • Different types of DNA damage may elicit different responses

  • Cell-type specificity may influence results (e.g., meristematic vs. differentiated cells)

  • Interactions with other pathways might vary under different conditions

What are the optimal conditions for CYCB1-1 antibody applications in Western blotting?

For optimal Western blotting results with CYCB1-1 antibody:

• Antibody dilution: Use a 1:1000 dilution of primary antibody for Western blotting applications .

• Expected molecular weight: Look for a band at approximately 55 kDa, which corresponds to the molecular weight of Cyclin B1 .

• Sample preparation considerations:

  • Include phosphatase inhibitors in extraction buffers to preserve phosphorylation states

  • Consider synchronizing cells/tissues to enrich for Cyclin B1, which is cell cycle-regulated

  • Include appropriate positive controls (tissues with known Cyclin B1 expression)

  • Use cycb1 mutants as negative controls to confirm antibody specificity

• Detection system optimization:

  • Use enhanced chemiluminescence (ECL) or fluorescent secondary antibodies

  • Adjust exposure times to prevent saturation while maintaining sensitivity

  • Consider stripping and reprobing with loading controls (tubulin, actin)

How should I optimize immunofluorescence protocols for CYCB1-1 detection?

For successful immunofluorescence detection of CYCB1-1:

• Antibody parameters:

  • Use a 1:400 dilution for immunofluorescence applications

  • Validate antibody specificity using appropriate controls (cycb1 mutants)

• Fixation optimization:

  • Test different fixatives (paraformaldehyde, methanol) to determine optimal antigen preservation

  • Optimize fixation duration and temperature

  • Consider antigen retrieval methods if signal is weak

• Signal enhancement strategies:

  • Use fluorophore-conjugated secondary antibodies with appropriate spectral properties

  • Consider tyramide signal amplification for weak signals

  • Optimize blocking solutions to reduce background (e.g., BSA, normal serum)

• Co-localization studies:

  • When performing double immunolabeling with DNA damage markers (e.g., γ-H2AX), carefully select antibodies raised in different species

  • Include appropriate single-labeling controls to assess cross-reactivity

  • Use confocal microscopy for precise co-localization analysis

What are the best approaches for validating CYCB1-1 antibody specificity?

Rigorous validation of CYCB1-1 antibody specificity is essential for reliable results:

• Genetic validation:

  • Compare antibody signal in wild-type versus cycb1 mutant tissues

  • For complete validation, use cycb1;1 cycb1;2 cycb1;3 cycb1;4 quadruple mutants if available

• Biochemical validation:

  • Perform peptide competition assays to confirm specificity

  • Use recombinant CYCB1-1 protein as a positive control

  • Conduct immunoprecipitation followed by mass spectrometry to confirm target identity

• Expression pattern correlation:

  • Compare antibody staining patterns with well-characterized CYCB1-1 promoter-reporter lines

  • Verify that the expression pattern matches expected cell cycle-regulated and DNA damage-induced patterns

• Cross-reactivity assessment:

  • Test antibody against multiple species to confirm cross-reactivity claims

  • The antibody should detect Cyclin B1 in Human, Mouse, Rat, Hamster, and Monkey samples as claimed

  • Verify that the detected protein has the expected molecular weight (55 kDa)