FBL17 Antibody

What is FBL17 and why is it important in plant research?

FBL17 (F-BOX-LIKE17) is an F-box protein in Arabidopsis thaliana that functions as a critical cell-cycle regulatory protein. It plays essential roles in pollen development and normal cell-cycle progression during the diploid sporophyte phase. FBL17 controls the stability of CYCLIN-DEPENDENT KINASE inhibitors called KIP-RELATED PROTEINs (KRPs), which explains the drastic reduction in cell division activity observed in both shoot and root apical meristems of fbl17 loss-of-function mutants . Beyond cell cycle regulation, FBL17 is also involved in DNA damage response (DDR) processes, with fbl17 mutants showing constitutive activation of DDR gene expression, higher frequency of DNA lesions, and increased cell death in root meristems even without genotoxic stress . Antibodies against FBL17 are therefore valuable tools for studying these fundamental biological processes.

What methods can be used to validate FBL17 antibody specificity?

To validate FBL17 antibody specificity, researchers should employ multiple complementary approaches:

• Western blot analysis: Compare wild-type Arabidopsis tissues with fbl17 mutant tissues. A specific antibody should show the predicted molecular weight band (~60 kDa) in wild-type samples that is absent or reduced in mutant samples .

• Immunofluorescence controls: Perform parallel immunostaining experiments using wild-type and fbl17 mutant tissues. Specific nuclear staining should be visible in wild-type but absent in mutant tissues .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before immunostaining or Western blotting. This should block specific binding and eliminate the signal.

• Antibody validation in transgenic lines: Test the antibody on tissues from transgenic lines expressing tagged FBL17 (such as FBL17-GFP) and confirm co-localization with anti-GFP antibody .

• Recombinant protein testing: Express recombinant FBL17 protein and confirm antibody binding through Western blot or ELISA.

What are the recommended fixation methods for FBL17 immunolocalization studies?

For optimal FBL17 immunolocalization in plant tissues, consider these fixation protocols:

• Paraformaldehyde fixation: Use 4% paraformaldehyde in PBS for 20-30 minutes at room temperature. This preserves protein antigenicity while maintaining cellular structure.

• Methanol fixation: For better nuclear protein detection, fix tissues in ice-cold methanol for 10 minutes, which removes lipids and dehydrates cells while precipitating proteins.

• Combination protocol: For detecting nuclear FBL17 foci, as described in the literature, use a combination approach: first fix with 4% paraformaldehyde (20 min), then post-fix with ice-cold methanol (10 min) .

• Permeabilization: After fixation, permeabilize tissues with 0.1-0.5% Triton X-100 in PBS to facilitate antibody access to nuclear proteins.

When studying FBL17 recruitment to DNA damage sites or co-localization with other proteins like γH2AX or RBR1, ensure that the fixation method preserves nuclear architecture and protein-protein interactions .

What are typical expression patterns of FBL17 in plant tissues?

FBL17 shows distinctive expression patterns in Arabidopsis tissues:

• Cell-type specificity: FBL17 is expressed in a restricted subset of cells in the root meristem .

• Cell cycle dependency: FBL17 expression follows a cell cycle phase-dependent pattern, which should be considered when designing experiments to detect the protein .

• Subcellular localization: FBL17 is primarily a nuclear F-box protein, as demonstrated in studies using FBL17-GFP reporter lines .

• Response to DNA damage: Upon zeocin treatment (which induces DNA double-strand breaks), FBL17 forms nuclear foci that co-localize with γH2AX, a marker of DNA lesion sites .

• Co-localization with cell cycle regulators: FBL17 co-localizes with RETINOBLASTOMA RELATED1 (RBR1) protein, particularly at sites of DNA damage .

Researchers should consider these patterns when planning immunostaining experiments and interpreting antibody-based detection results.

How can FBL17 antibodies be used to study DNA damage response mechanisms?

FBL17 antibodies provide powerful tools for investigating DNA damage response (DDR) mechanisms in plants:

• Immunofluorescence co-localization studies: Use anti-FBL17 antibodies together with antibodies against DDR markers (like γH2AX) to study recruitment of FBL17 to DNA damage sites. Quantitative analysis of co-localization can reveal temporal dynamics of protein recruitment .

• Chromatin immunoprecipitation (ChIP): Apply FBL17 antibodies in ChIP experiments to identify genomic regions where FBL17 is recruited following DNA damage, potentially in association with RBR1/E2FA complexes .

• Proximity ligation assay (PLA): Combine FBL17 antibodies with antibodies against RBR1, γH2AX, or other DDR proteins to visualize and quantify protein-protein interactions at DNA damage sites with single-molecule resolution.

• Time-course experiments: Use immunofluorescence with FBL17 antibodies to track the temporal dynamics of FBL17 recruitment after treatment with different DNA-damaging agents (zeocin for DSBs, cisplatin for crosslinks, or hydroxyurea for replication stress) .

• Co-immunoprecipitation (Co-IP): Apply FBL17 antibodies in Co-IP experiments to pull down protein complexes and identify binding partners specific to different DNA damage contexts.

Research has shown that FBL17 is specifically recruited to nuclear foci upon zeocin treatment (causing double-strand breaks) but not after cisplatin or hydroxyurea treatments, suggesting specificity to certain types of DNA damage .

What controls should be included when using FBL17 antibodies in co-localization experiments?

When performing co-localization experiments with FBL17 antibodies, include these essential controls:

• Single antibody controls: Perform staining with each antibody individually to ensure signal specificity and absence of bleed-through between fluorescence channels.

• Genetic controls: Include fbl17 mutant samples as negative controls to confirm antibody specificity .

• Treatment controls: Compare untreated samples with genotoxic stress-treated samples (e.g., zeocin, cisplatin, hydroxyurea) to establish baseline versus induced localization patterns .

• Antibody cross-reactivity controls: Test secondary antibodies alone to ensure they don't bind non-specifically to the sample.

• Random co-localization assessment: Perform statistical analysis (e.g., Pearson's correlation coefficient) to distinguish true co-localization from random overlap.

• Spatial resolution controls: Include proteins known not to co-localize with FBL17 to verify the resolving power of your microscopy setup.

Quantitative analysis should be conducted, as demonstrated in the literature where researchers found approximately 5% co-localization of RBR1 with γH2AX and 5% co-localization of FBL17 with RBR1 after zeocin treatment, with only 1% showing co-localization of all three proteins together .

How can FBL17 antibodies be optimized for Western blot analysis of plant tissues?

For optimal Western blot detection of FBL17 in plant tissues:

• Protein extraction optimization:

  • Use a nuclear extraction protocol since FBL17 is a nuclear protein

  • Include proteasome inhibitors (e.g., MG132) to prevent degradation of this F-box protein

  • Add phosphatase inhibitors to preserve potential post-translational modifications

• Sample preparation:

  • Synchronize plant cell populations when possible, as FBL17 shows cell cycle-dependent expression

  • When studying DNA damage responses, compare samples with and without genotoxic treatments

• Gel electrophoresis parameters:

  • Use 10-12% SDS-PAGE gels for optimal resolution of FBL17 (~60 kDa)

  • Consider running parallel gels with phospho-specific antibodies if phosphorylation status is relevant

• Antibody incubation optimization:

  • Test different antibody dilutions (typically 1:500 to 1:2000)

  • Optimize incubation time and temperature (4°C overnight often yields best results)

  • Use 5% BSA instead of milk for blocking when detecting phosphorylated versions of FBL17

• Signal enhancement strategies:

  • Consider using enhanced chemiluminescence (ECL) substrates for greater sensitivity

  • For low-abundance detection, try biotin-streptavidin amplification systems

• Validation approaches:

  • Always include wild-type and fbl17 mutant samples as positive and negative controls

  • Consider using FBL17-GFP transgenic lines and detecting with both anti-FBL17 and anti-GFP antibodies

What considerations are important when studying FBL17 interactions with RBR1 and E2FA?

When investigating FBL17 interactions with RBR1 and E2FA, researchers should consider:

• Experimental design factors:

  • Cell cycle stage is crucial, as these interactions may be cell cycle-dependent

  • DNA damage conditions significantly affect these interactions, with zeocin treatment particularly relevant

  • Use appropriate fixation methods that preserve protein complexes

• Methodological approaches:

  • Co-immunoprecipitation with antibodies against FBL17, RBR1, and E2FA

  • Bimolecular Fluorescence Complementation (BiFC) to visualize interactions in vivo

  • Proximity Ligation Assay (PLA) for high-resolution detection of protein-protein interactions

  • FRET/FLIM analysis when using fluorescent protein fusions

• Controls and validations:

  • Use protein interaction domain mutants as negative controls

  • Verify interactions with multiple techniques

  • Include cell cycle phase markers to correlate interactions with cell cycle stages

• Functional analysis:

  • Compare wild-type with fbl17, rbr1, and e2fa mutant backgrounds

  • Assess the effect of FBL17 depletion on RBR1/E2FA target gene expression

  • Evaluate DNA damage response in different genetic backgrounds

Research has shown that FBL17 nuclear foci co-localize with RBR1 after zeocin treatment, and importantly, FBL17 and γH2AX never co-localize if RBR1 is not present at these foci, suggesting that RBR1 mediates the recruitment of FBL17 to DNA damage sites .

What are common issues when detecting FBL17 in immunostaining experiments and how can they be resolved?

Researchers frequently encounter these challenges when using FBL17 antibodies for immunostaining:

• Low signal intensity:

  • Cause: Insufficient antibody concentration or low FBL17 expression

  • Solution: Increase antibody concentration, extend incubation time, or use signal amplification systems like tyramide signal amplification

• High background:

  • Cause: Non-specific binding or excessive antibody concentration

  • Solution: Optimize blocking (try 5% BSA or goat serum), increase washing steps, and titrate antibody concentration

• Inconsistent nuclear foci detection:

  • Cause: Cell cycle-dependent expression of FBL17 or variation in DNA damage levels

  • Solution: Synchronize cell populations when possible and carefully control genotoxic treatments

• Poor co-localization with DDR markers:

  • Cause: Temporal dynamics of recruitment or fixation issues

  • Solution: Perform time-course experiments after DNA damage induction and optimize fixation protocols

• Low frequency of nuclei with FBL17 foci:

  • Cause: As reported in literature, only a subset of nuclei show FBL17 foci after zeocin treatment

  • Solution: Increase sampling size and quantify foci systematically across multiple biological replicates

• Fixation-related antigen masking:

  • Cause: Certain fixatives may mask FBL17 epitopes

  • Solution: Test different fixation protocols or include an antigen retrieval step

How can FBL17 antibodies be used to investigate the relationship between cell cycle regulation and DNA damage response?

FBL17 antibodies offer valuable tools to explore the intersection of cell cycle control and DNA damage response:

• Dual immunolabeling approaches:

  • Combine FBL17 antibodies with markers for specific cell cycle phases (e.g., EdU for S-phase, CYCB1;1 for G2/M)

  • Co-stain for both FBL17 and DDR markers like γH2AX to correlate cell cycle position with DNA damage

• Experimental design strategies:

  • Compare FBL17 localization in synchronized cell populations at different cell cycle stages

  • Assess how DNA damage affects FBL17 localization across the cell cycle

  • Analyze FBL17 recruitment to damage sites in cells arrested at specific cell cycle stages

• Genetic approach combinations:

  • Use FBL17 antibodies in different genetic backgrounds (e.g., krp2 mutants, e2fa mutants)

  • Compare DDR gene expression (using RT-qPCR) with FBL17 protein levels/localization

• Quantitative analysis methods:

  • Measure the frequency of FBL17-γH2AX co-localization across different cell cycle phases

  • Quantify nuclear FBL17 levels relative to DDR activation markers

  • Track temporal dynamics of FBL17 recruitment to damage sites throughout the cell cycle

Research has shown that fbl17 mutants exhibit constitutive upregulation of DDR genes that is not observed in KRP2 overexpression lines, suggesting that FBL17's role in DDR is independent of its cell cycle regulatory function through KRP degradation .

What protocols are recommended for ChIP experiments using FBL17 antibodies?

For successful Chromatin Immunoprecipitation (ChIP) experiments with FBL17 antibodies:

• Sample preparation:

  • Use 1-2g of Arabidopsis seedlings (10-14 days old)

  • Consider treatments with DNA-damaging agents like zeocin to induce FBL17 recruitment to damage sites

  • Crosslink with 1% formaldehyde for 10 minutes at room temperature

• Chromatin extraction and processing:

  • Isolate nuclei before sonication to enrich for nuclear FBL17

  • Optimize sonication conditions to generate 200-500bp DNA fragments

  • Pre-clear chromatin with protein A/G beads to reduce background

• Immunoprecipitation strategy:

  • Use 2-5μg of anti-FBL17 antibody per IP reaction

  • Include IgG control and input samples

  • Consider dual ChIP approaches with RBR1 antibodies to identify co-occupied regions

• Washing and elution optimization:

  • Use stringent washing conditions (increasing salt concentrations)

  • Elute DNA-protein complexes at 65°C to improve recovery

• Data analysis considerations:

  • Design primers targeting promoters of DDR genes shown to be upregulated in fbl17 mutants

  • Include regions known to be bound by RBR1/E2FA complexes as suggested by previous studies

  • Compare binding patterns before and after DNA damage induction

• Validation approaches:

  • Perform ChIP-qPCR on key target genes before proceeding to ChIP-seq

  • Verify findings with alternative approaches such as DNA-protein interaction assays

How can different antibody-based techniques be combined to study FBL17 function in DNA repair?

A multi-technique approach using FBL17 antibodies can provide comprehensive insights into its DNA repair functions:

• Technique integration strategy:

  Technique	Application	Key Insight
  Immunofluorescence	Localization and dynamics	FBL17 recruitment to DNA damage foci
  Co-immunoprecipitation	Protein interaction partners	FBL17 associations with RBR1 and repair proteins
  ChIP-seq	Genomic binding sites	Direct or indirect DNA interactions
  Proximity ligation assay	In situ protein interactions	Spatial organization of repair complexes
  Western blotting	Protein levels and modifications	DNA damage-induced changes in FBL17

• Sequential experimental approach:

  • Begin with immunofluorescence to establish basic localization patterns after DNA damage

  • Follow with co-IP to identify binding partners specific to damage conditions

  • Perform ChIP-seq to determine genomic recruitment sites

  • Use proximity ligation to confirm specific interactions at damage sites

  • Monitor protein modifications with phospho-specific antibodies

• Integrative data analysis:

  • Correlate FBL17 binding sites with transcriptional changes in fbl17 mutants

  • Map the temporal sequence of protein recruitment to damage sites

  • Identify DNA repair pathways specifically affected by FBL17 activity

Research has shown that FBL17 is specifically recruited to DNA double-strand break sites (marked by γH2AX) but only in the presence of RBR1, suggesting a coordinated recruitment mechanism that could be further explored through these integrated approaches .

How might phospho-specific FBL17 antibodies advance our understanding of its regulation?

Developing and utilizing phospho-specific FBL17 antibodies could significantly advance the field:

• Potential phosphorylation sites:

  • Kinase prediction algorithms suggest potential ATM/ATR target sites in FBL17

  • Phosphorylation may regulate FBL17 stability, localization, or substrate binding

• Experimental applications:

  • Phospho-specific antibodies could track DNA damage-induced modifications of FBL17

  • Western blot analysis with phospho-specific antibodies before and after DNA damage treatment

  • Immunoprecipitation with phospho-specific antibodies to identify interactors specific to phosphorylated FBL17

• Research questions to address:

  • Does DNA damage trigger phosphorylation of FBL17?

  • Is FBL17 phosphorylation required for its recruitment to DNA damage sites?

  • Do different types of DNA damage induce distinct phosphorylation patterns?

  • Which kinases are responsible for FBL17 phosphorylation in response to damage?

• Methodological considerations:

  • Validate phospho-specific antibodies using phosphatase treatments

  • Use phospho-mimetic and phospho-dead FBL17 mutants as controls

  • Consider mass spectrometry approaches to identify all phosphorylation sites

What protocols can be used to study FBL17 substrates in the context of DNA damage?

To identify and characterize FBL17 substrates involved in DNA damage response:

• Immunoprecipitation-based approaches:

  • Use anti-FBL17 antibodies for co-IP experiments followed by mass spectrometry

  • Compare binding partners before and after DNA damage treatments

  • Conduct reciprocal IPs to confirm interactions

• Degradation assays:

  • Develop in vitro ubiquitination assays using immunopurified FBL17 complexes

  • Monitor protein stability of candidate substrates in wild-type versus fbl17 mutant backgrounds

  • Use proteasome inhibitors to confirm ubiquitin-mediated degradation

• Proximity-based labeling:

  • Create FBL17-BioID or FBL17-APEX2 fusion proteins

  • Induce proximity labeling before and after DNA damage

  • Identify biotinylated proteins by streptavidin pulldown and mass spectrometry

• Genetic interaction studies:

  • Test genetic interactions between fbl17 and mutations in candidate substrate genes

  • Look for suppression or enhancement of DNA damage sensitivity phenotypes

• Domain mapping experiments:

  • Use FBL17 antibodies with truncated FBL17 variants to map substrate binding domains

  • Perform competition assays with peptides derived from potential substrates

Research has shown that while KRP2 is a known substrate of FBL17 in cell cycle regulation, KRP2 overexpression does not recapitulate the DDR phenotypes of fbl17 mutants, suggesting the existence of additional substrates specific to DNA damage contexts .

How can super-resolution microscopy enhance FBL17 antibody-based studies?

Super-resolution microscopy offers powerful new capabilities for FBL17 antibody applications:

• Technical advantages for FBL17 research:

  • Resolve individual FBL17 foci beyond the diffraction limit (typically ~250nm)

  • Precisely measure co-localization with RBR1, γH2AX, and other DNA repair factors

  • Determine the exact spatial organization of FBL17 within nuclear repair foci

• Applicable super-resolution techniques:

  • STORM/PALM: Single-molecule localization microscopy for highest spatial resolution

  • SIM: Structured illumination microscopy for live-cell compatibility

  • STED: Stimulated emission depletion for detailed nuclear architecture

• Experimental design considerations:

  • Use secondary antibodies coupled to photoswitchable fluorophores for STORM

  • Optimize fixation protocols to minimize background fluorescence

  • Design multi-color experiments to visualize FBL17 alongside RBR1 and γH2AX

• Quantitative analysis approaches:

  • Measure precise distances between FBL17 and other repair factors

  • Determine the size and molecular composition of FBL17-containing foci

  • Track temporal dynamics of FBL17 recruitment with nanometer precision

• Validation strategies:

  • Compare results across multiple super-resolution modalities

  • Correlate with conventional confocal microscopy data

  • Verify biological findings with complementary biochemical approaches

Super-resolution could help resolve the finding that only approximately 1% of nuclear foci show co-localization of FBL17, RBR1, and γH2AX together, potentially revealing more subtle interaction dynamics not visible with conventional microscopy .

What considerations are important when developing new monoclonal antibodies against FBL17?

For researchers developing new monoclonal antibodies against FBL17:

• Epitope selection strategy:

  • Target unique regions of FBL17 that don't share homology with other F-box proteins

  • Consider epitopes outside the F-box domain for specificity

  • Avoid regions involved in protein-protein interactions that might be masked in complexes

  • Create separate antibodies recognizing different domains (N-terminal, F-box, C-terminal)

• Immunization and screening approach:

  • Use both peptide antigens and recombinant protein fragments

  • Screen hybridomas with both wild-type and fbl17 mutant protein extracts

  • Test antibody performance in multiple applications (WB, IP, IF, ChIP)

• Validation requirements:

  • Confirm specificity using knockout/knockdown controls

  • Verify recognition of native and denatured FBL17

  • Test cross-reactivity with related proteins

  • Evaluate specificity across different plant species if cross-reactivity is desired

• Application-specific optimization:

  • For immunofluorescence: Test different fixation and permeabilization methods

  • For ChIP: Validate chromatin binding capacity

  • For Western blot: Optimize for reducing and non-reducing conditions

• Considerations for phospho-specific antibodies:

  • Identify likely phosphorylation sites through predictive algorithms

  • Consider sites that might be modified during DNA damage response

  • Use phospho-peptides for immunization and non-phosphorylated peptides for counter-screening

How do experimental approaches for studying FBL17 differ between plant and mammalian systems?

Although FBL17 is primarily studied in plants, comparative approaches with mammalian systems offer valuable insights:

• System-specific considerations:

  Aspect	Plant Systems (Arabidopsis)	Mammalian Systems
  Genetic tools	T-DNA insertion mutants, CRISPR	siRNA, shRNA, CRISPR
  Cell culture	Plant cell suspensions, protoplasts	Established cell lines
  Visualization	Whole-mount roots, transgenic reporters	Cell monolayers, tissue sections
  DNA damage induction	Zeocin, cisplatin, hydroxyurea	Similar agents, plus IR, UV
  Antibody availability	Limited commercial options	More extensive commercial options

• Functional homology considerations:

  • Plant FBL17 and mammalian SKP2 share some functional similarities as cell cycle regulators

  • Both participate in SCF complexes targeting cell cycle inhibitors for degradation

  • DNA damage response roles may show convergent evolution

• Experimental design adaptations:

  • Mammalian studies often use synchronized cell cultures, which can be adapted for plant systems

  • Immunoprecipitation protocols require different buffer optimizations between systems

  • Microscopy preparations differ significantly between adherent animal cells and plant tissues

• Cross-system validation strategies:

  • Test if mammalian antibodies against functionally similar proteins cross-react with plant FBL17

  • Compare FBL17 recruitment kinetics to damage sites with those of mammalian counterparts

  • Assess functional complementation between systems

Understanding these differences is essential when interpreting results across systems or adapting protocols from mammalian to plant research contexts.

What are the key considerations when designing experiments to study FBL17 recruitment to DNA damage sites?

When investigating FBL17 recruitment to DNA damage sites, researchers should consider:

• DNA damage induction approaches:

  • Agent selection: Zeocin specifically induces FBL17 foci formation, while cisplatin and hydroxyurea do not

  • Dose optimization: Titrate doses to ensure cell viability while inducing sufficient damage

  • Temporal dynamics: Perform time-course experiments (15min to 24h) to capture recruitment kinetics

• Experimental controls:

  • Genetic controls: Include fbl17 mutants as negative controls

  • Treatment controls: Compare untreated samples for baseline localization

  • Co-localization controls: Include γH2AX staining to confirm DNA damage sites

• Visualization strategy:

  • Live imaging options: Consider FBL17-fluorescent protein fusions for dynamic studies

  • Fixed sample approaches: Optimize fixation for simultaneous detection of FBL17, RBR1, and γH2AX

  • Resolution requirements: Super-resolution techniques may be necessary to resolve fine structures

• Quantification methods:

  • Focus counting: Determine number and size of FBL17 foci per nucleus

  • Co-localization analysis: Measure overlap coefficients with DNA damage markers

  • Recruitment kinetics: Track appearance and disappearance of foci over time

• Dependency testing:

  • Genetic dependencies: Test requirement for RBR1, ATM/ATR kinases, and repair factors

  • Protein domain requirements: Use truncated versions of FBL17 to map recruitment domains

  • Post-translational modifications: Investigate role of phosphorylation in recruitment

Research has shown that FBL17 recruitment to γH2AX foci depends on RBR1, as they never co-localize if RBR1 is not present at these foci , suggesting complex regulation of this process.

How can researchers distinguish between FBL17's cell cycle and DNA damage response functions?

To differentiate between FBL17's dual roles in cell cycle regulation and DNA damage response:

• Genetic separation of function approaches:

  • Study FBL17 domain mutants that might separate different functions

  • Compare phenotypes of fbl17 mutants with krp2 mutants and KRP2 overexpressors

  • Create separation-of-function mutants through targeted mutagenesis

• Substrate identification strategies:

  • Identify FBL17 substrates specific to DNA damage contexts versus cell cycle contexts

  • Compare ubiquitination targets before and after DNA damage

  • Look for substrates that do not accumulate in KRP2 overexpression lines

• Temporal analysis methods:

  • Synchronize cells and study FBL17 function across different cell cycle phases

  • Determine if FBL17's DDR function is restricted to specific cell cycle phases

  • Use time-lapse imaging with cell cycle phase markers alongside FBL17

• Expression analysis approaches:

  • Compare transcriptional profiles of fbl17 mutants with KRP2 overexpressors

  • Focus on genes specifically misregulated in fbl17 but not in KRP2 overexpressors

  • Perform ChIP-seq to identify direct targets of FBL17-containing complexes

• Functional assays to differentiate roles:

  • Measure DNA repair efficiency in various genetic backgrounds

  • Assess cell cycle progression independently from DNA damage induction

  • Evaluate replication stress response separately from DSB repair capacity

Research has shown that KRP2 overexpression lines do not exhibit the constitutive DDR gene upregulation seen in fbl17 mutants, providing strong evidence that FBL17's role in DDR is not simply a consequence of its cell cycle function through KRP regulation .