CCND3 Antibody

What is CCND3 and what roles does it play in cellular function?

Cyclin D3 (CCND3) is a member of the D-type cyclins, which are key regulators of Cyclin-dependent kinases 4 and 6 (CDK4/6). It mediates growth factor-induced progression through the G1 phase in the cell cycle. CCND3 functions through:

• Forming a complex with CDK4/6 to phosphorylate retinoblastoma (RB) protein family members

• Regulating G1/S phase transition by allowing dissociation of E2F transcription factors from RB/E2F complexes

• Contributing to both CDK-dependent cell cycle progression and CDK-independent transcriptional activation

• Participating in metabolic control mechanisms

CCND3 is widely expressed across tissues but shows differential importance in specific cell types, with particular significance in lymphoid cells and certain tumor types. Research indicates CCND3 may have distinct functions from other D-type cyclins in tissues such as germinal center B cells, where it appears to be indispensable for proper development and function .

What are the molecular characteristics of the CCND3 protein?

The CCND3 protein has several key molecular characteristics:

The protein contains several functional regions including CDK-binding domains and phosphorylation sites important for its regulation and activity .

What types of antibodies are available for CCND3 detection?

Researchers have access to multiple types of CCND3 antibodies with different characteristics:

When selecting an antibody, researchers should consider:

• The specific application requirements (WB, IHC, Flow, etc.)

• Species reactivity needed (human, mouse, rat, etc.)

• Whether detection of post-translational modifications is required

• Clone specificity for targeted epitopes

How should I optimize Western blot protocols for CCND3 detection?

For optimal Western blot detection of CCND3, consider the following methodology:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors

  • Include phosphatase inhibitors if phosphorylated forms are of interest

  • Typical protein loading: 20-50 μg of total protein per lane

• Gel selection and transfer:

  • 10-12% SDS-PAGE gels are appropriate for resolving the 33-36 kDa CCND3 protein

  • Standard PVDF or nitrocellulose membranes are suitable for transfer

• Antibody dilutions (optimize for each specific antibody):

  • Primary antibody: Typically 1:500-1:2000 for polyclonal antibodies

  • For monoclonal antibodies, follow manufacturer recommendations

  • Secondary antibody: Usually 1:5000-1:10000

• Visualization methods:

  • Both enhanced chemiluminescence (ECL) and fluorescent detection methods work well

  • For quantitative analysis, consider fluorescent secondaries

• Controls to include:

  • Positive control: Cell lines known to express CCND3 (many tumor cell lines)

  • Negative control: CCND3 knockout or knockdown samples if available

  • Loading control: Beta-actin, GAPDH, or other housekeeping proteins

Troubleshooting: If multiple bands appear, this may indicate degradation products or post-translational modifications. Freshly prepared samples and inclusion of appropriate inhibitors can help minimize these issues .

What are best practices for immunohistochemistry using CCND3 antibodies?

For effective immunohistochemical detection of CCND3:

• Fixation and sectioning:

  • Formalin-fixed paraffin-embedded (FFPE) sections: Use standard 10% neutral buffered formalin

  • Frozen sections: Fix with 4% paraformaldehyde or acetone

  • Section thickness: 4-5 μm recommended

• Antigen retrieval (critical step):

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) works well for most CCND3 antibodies

  • Pressure cooker/microwave methods typically yield best results

• Blocking and antibody incubations:

  • Block with 5-10% normal serum from secondary antibody host species

  • Primary antibody dilutions: 1:50-1:200 for paraffin sections

  • Incubation times: Overnight at 4°C often yields best results

  • Secondary detection: Both HRP/DAB and fluorescent methods are suitable

• Signal development and counterstaining:

  • For chromogenic detection: 3,3'-Diaminobenzidine (DAB) substrate is commonly used

  • Counterstain nuclei with hematoxylin for brightfield or DAPI for fluorescence

  • Expected pattern: Predominantly nuclear and some cytoplasmic staining

• Controls:

  • Positive control: Lymphoid tissues or tumor samples with known CCND3 expression

  • Negative control: Omit primary antibody or use isotype control

Note that detection sensitivity can vary between antibody clones and fixation methods. Optimization may be required when working with new tissue types or antibody batches .

How do I properly validate a CCND3 antibody for my research?

Thorough validation of CCND3 antibodies should include:

• Specificity testing:

  • Western blot showing a band of expected molecular weight (33-36 kDa)

  • Peptide competition assay to confirm specific binding

  • Testing in CCND3 knockout/knockdown systems as negative controls

  • Comparison with alternative antibody clones targeting different epitopes

• Application-specific validation:

  • For IHC: Compare staining patterns with published literature

  • For flow cytometry: Use fluorescence-minus-one (FMO) controls

  • For IF/ICC: Colocalize with other markers of known subcellular distribution

• Cross-reactivity assessment:

  • Test for potential cross-reactivity with other cyclin family members (especially CCND1 and CCND2)

  • Confirm species specificity if working across multiple models

• Reproducibility testing:

  • Test multiple lots if available

  • Establish consistent protocols with defined positive controls

• Publication verification:

  • Check if the specific antibody has been cited in peer-reviewed publications

  • Review any associated validation data from manufacturers

Researchers should document all validation steps and consider reporting these in methods sections of publications to enhance reproducibility across the field .

How does CCND3 function in normal and malignant B cell development?

CCND3 plays critical roles in B cell development and lymphomagenesis:

• Germinal Center Formation:

  • CCND3 is essential for germinal center (GC) B cell proliferation

  • Ccnd3-/- mice show profound defects in GC formation and maintenance

  • GC B cells specifically upregulate CCND3 compared to other D-type cyclins

• Antibody Responses:

  • Mice lacking CCND3 show impaired T-dependent antibody responses

  • Lower serum anti-NP titers observed across most IgG subclasses

  • Defects in affinity maturation during primary responses, though repeated immunization can partially overcome this deficiency

• B-cell Malignancies:

  • CCND3 is indispensable for maintenance of B-cell acute lymphoblastic leukemia (B-ALL)

  • Knockdown of CCND3 induces apoptosis in B-ALL cells regardless of underlying driver mutations

  • CCND3 contributes to CDK8 transcription, which may explain its anti-apoptotic effect

• Recurrent Mutations:

  • Recurrent mutations in CCND3 have been identified in MLL-rearranged acute myeloid leukemia

  • These mutations may drive disease progression through altered cell cycle regulation

These findings highlight CCND3 as a potential therapeutic target in B-cell malignancies, with distinct functions that cannot be compensated by other D-type cyclins in certain contexts .

What is the relationship between CCND3 expression and CDK4/6 inhibitor resistance?

Research has revealed important connections between CCND3 and resistance to CDK4/6 inhibitors:

• Upregulation in Response to Treatment:

  • Short-term treatment with palbociclib (a CDK4/6 inhibitor) strongly increases CCND3 protein expression in B-ALL cell lines

  • This upregulation appears to be an adaptive resistance mechanism

• Experimental Evidence:

  • Ectopic CCND3 overexpression significantly decreases sensitivity of B-ALL cells to palbociclib

  • B-ALL cells selected for palbociclib resistance show dramatically elevated CCND3 expression levels

  • Palbociclib-resistant cells remain sensitive to CCND3 knockdown, suggesting CCND3 dependency persists

• Mechanism of Action:

  • The anti-apoptotic effect of CCND3 appears independent of CDK4/6 kinase activity

  • CCND3 contributes to CDK8 transcription, potentially explaining some of its CDK4/6-independent functions

  • Combined targeting of CCND3 expression rather than just CDK4/6 activity may provide superior therapeutic outcomes

• Clinical Implications:

  • Monitoring CCND3 expression levels may help predict response to CDK4/6 inhibitors

  • Targeting CCND3 directly might overcome resistance to CDK4/6 inhibitors in certain malignancies

  • Combinatorial approaches targeting both CCND3 expression and CDK4/6 activity could potentially enhance therapeutic efficacy

These findings suggest that strategies aimed at downregulating CCND3 expression might be superior to inhibition of CDK4/6 kinase activity alone in certain treatment contexts.

How does CCND3 influence erythrocyte development and characteristics?

CCND3 plays an unexpected but critical role in regulating erythrocyte size and number:

• Genetic Association:

  • Human genetic studies identified variants in an erythroid-specific enhancer of CCND3 that associate with mean corpuscular volume (MCV) and red blood cell (RBC) count

  • Individuals with weaker enhancer variants have larger but fewer red cells

• Knockout Mouse Phenotype:

  • Ccnd3 knockout mice display:

    • 38% reduction in RBC count

    • 40% increase in erythrocyte size (MCV)

    • 13% reduction in hematocrit

    • These mice have the largest adult mouse erythrocytes described to date, approaching human RBC size

• Mechanism of Action:

  • CCND3 regulates the number of cell divisions that erythroid precursors undergo before terminal differentiation

  • Fewer divisions result in larger but fewer erythrocytes

  • This reveals an unexpected link between cell cycle regulation and erythrocyte production

• Research Applications:

  • CCND3 antibodies can be used to study erythropoiesis and erythrocyte size control

  • Immunostaining of bone marrow or flow cytometry to detect CCND3 in erythroid precursors can provide insights into erythrocyte developmental abnormalities

  • Useful for investigating macrocytic anemias and other RBC disorders

This research highlights how cell cycle regulators like CCND3 can have specialized tissue-specific functions beyond their canonical roles, linking fundamental cell biology to physiological outcomes.

What are the recommended experimental approaches for studying CCND3 phosphorylation?

CCND3 phosphorylation is a critical regulatory mechanism affecting its function, stability, and interactions. Here are recommended approaches for studying these modifications:

• Detection of phosphorylated forms:

  • Phospho-specific antibodies targeting key sites (e.g., Thr283) are available for western blot and ELISA applications

  • Phos-tag™ SDS-PAGE can resolve phosphorylated from non-phosphorylated species

  • Mass spectrometry for unbiased identification of all phosphorylation sites

• Functional analysis approaches:

  • Site-directed mutagenesis of phosphorylation sites (Thr→Ala to prevent; Thr→Asp to mimic)

  • Expression of phospho-mutants to assess:

    • Protein stability and half-life

    • CDK binding affinity

    • Subcellular localization

    • Cell cycle progression effects

• Kinase identification:

  • In vitro kinase assays with recombinant CCND3 protein

  • Small molecule kinase inhibitors to identify responsible pathways

  • Kinase overexpression/knockdown to observe effects on CCND3 phosphorylation

• Phosphorylation dynamics:

  • Cell synchronization to track phosphorylation changes throughout cell cycle

  • Growth factor stimulation/deprivation to assess signaling-dependent modifications

  • Monitor effects of cellular stress or differentiation signals on phosphorylation status

• Protein-protein interaction studies:

  • Co-immunoprecipitation comparing wild-type vs. phospho-mutants

  • Proximity ligation assays to detect interactions in situ

  • Phosphorylation-dependent binding partner identification using mass spectrometry

Known phosphorylation sites on CCND3 include T9, T261, S263, and S264, with varying effects on protein function and stability . Researchers should consider using multiple complementary approaches to thoroughly characterize the functional significance of specific phosphorylation events.

How can CCND3 antibodies be employed to study its non-canonical functions?

Beyond its classical role in cell cycle regulation, CCND3 exhibits several non-canonical functions that can be investigated using specialized antibody-based approaches:

• Transcriptional co-activation:

  • Chromatin immunoprecipitation (ChIP) using CCND3 antibodies to identify genomic binding sites

  • Sequential ChIP (ChIP-reChIP) to detect co-occupancy with transcription factors like ATF5

  • RNA-seq following CCND3 knockdown to identify CDK-independent transcriptional targets

• CDK-independent protein interactions:

  • Co-immunoprecipitation with CCND3 antibodies followed by mass spectrometry

  • Proximity ligation assays to visualize interactions in situ

  • FRET/BRET approaches using tagged proteins to monitor direct interactions

• Metabolic functions:

  • Immunofluorescence co-localization with metabolic enzymes or organelle markers

  • Fractionation studies with subsequent immunoblotting for CCND3

  • Metabolomic analysis following CCND3 manipulation (overexpression/knockdown)

• Tissue-specific roles:

  • Immunohistochemistry across diverse tissues to identify unique expression patterns

  • Single-cell analysis using flow cytometry with CCND3 antibodies

  • Studies in specialized cell types like germinal center B cells where CCND3 has critical functions

• Disease-specific applications:

  • Analysis of CCND3 in patient samples using IHC to correlate with clinical outcomes

  • Monitoring CCND3 expression in drug resistance development

  • Detection of mutant forms using specialized antibodies when available

When investigating non-canonical functions, researchers should carefully validate antibody specificity in the particular experimental system and consider using multiple antibody clones targeting different epitopes to confirm findings.