CC2D1A Antibody

What is CC2D1A and what functional domains does it contain?

CC2D1A is a 104.06 kDa protein containing several distinct domains. It features multiple DM14 domains and a C2 domain, which are critical for its structural integrity and functional roles. The C2 domain is involved in calcium-dependent phospholipid binding, while the DM14 domains are unique to the CC2D1A protein family with functions that remain under investigation . CC2D1A functions as both a transcription factor that binds specifically with the dual repressor element in neuronal cells and as a cytoplasmic signaling scaffold protein .

What are the primary expression patterns of CC2D1A in mammalian tissues?

CC2D1A is predominantly expressed in the brain, particularly during embryonic development, indicating its importance in neurodevelopmental processes . Within the brain, highest expression is observed in the cerebral cortex and hippocampus. Double immunofluorescence staining has confirmed that CC2D1A is expressed in both excitatory and inhibitory neurons of the adult hippocampus . Additionally, CC2D1A is expressed in ciliated tissues, including the left-right organizer, epidermis, pronephric duct, nephrostomes, and ventricular zone of the brain .

What disease associations have been established for CC2D1A mutations?

CC2D1A mutations have been implicated in a spectrum of conditions including:

• Autosomal recessive non-syndromic mental retardation (NSMR)

• Autism spectrum disorder (ASD)

• Intellectual disability

• Seizures

• Heterotaxy syndrome and ciliary dysfunction

• Renal dysplasia and cystic kidneys

Notably, a protein truncating mutation in CC2D1A has been identified in nine consanguineous families with severe autosomal recessive NSMR .

What types of CC2D1A antibodies are available for research applications?

Several types of CC2D1A antibodies have been developed for research purposes:

Both antibody types show specific immunoreactivity to endogenous CC2D1A in cancer cell lines and tissue samples .

How should I validate a CC2D1A antibody for my specific experimental system?

Validation of CC2D1A antibodies should follow these methodological steps:

• Western blot analysis: Use positive control lysates from tissues known to express CC2D1A (brain tissue or neuronal cell lines) alongside negative controls such as CC2D1A knockout cells. A validated antibody should detect a single band at approximately 104 kDa .

• Cross-validation: Compare results with multiple antibodies targeting different epitopes of CC2D1A (e.g., N-terminal and C-terminal regions) to confirm specificity .

• Immunoprecipitation followed by mass spectrometry: This confirms that the antibody is pulling down the correct protein.

• Immunohistochemistry controls: Use serial dilutions of CC2D1A antibody until a reproducible pattern of staining intensity emerges (1:50 dilution has been reported as effective) .

• Knockout/knockdown verification: The absence of signal in CC2D1A knockout/knockdown samples provides definitive validation. This approach confirmed antibody specificity in patient-derived lymphoblastoid cells where western blot analysis showed that antibodies identified a 104 kDa band in normal individuals but not in patients with CC2D1A mutations .

How can I optimize Western blot protocols for CC2D1A detection?

For optimal Western blot detection of CC2D1A, follow these methodological recommendations:

• Sample preparation: Extract proteins using RIPA buffer supplemented with protease inhibitors. For brain tissue samples, use specialized neural protein extraction reagents.

• Gel selection: Use 8% SDS-PAGE gels to achieve good resolution for the 104 kDa CC2D1A protein.

• Transfer conditions: Transfer proteins to PVDF membranes at 100V for 90 minutes in cold transfer buffer containing 20% methanol.

• Blocking conditions: Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Antibody dilutions: For primary antibodies, optimal dilutions reported include:

  • N-terminus and C-terminus polyclonal antibodies: 1:1000 dilution

  • Monoclonal PCRP-CC2D1A-1C2: 1:500 dilution

• Incubation parameters: Incubate with primary antibody overnight at 4°C, followed by HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature.

• Detection method: Enhanced chemiluminescence (ECL) systems with exposure times of 1-5 minutes typically yield clear bands.

What immunohistochemistry protocols are recommended for CC2D1A localization studies?

For effective immunohistochemistry (IHC) of CC2D1A in tissue sections:

• Tissue preparation: Fix tissues in 4% paraformaldehyde and embed in paraffin. Cut sections at 5-7 μm thickness.

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes.

• Blocking: Block endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes, followed by protein blocking with 5% normal serum.

• Antibody application: Apply CC2D1A antibody at 1:50 dilution and incubate overnight at 4°C .

• Detection system: Use biotin-streptavidin-HRP detection systems followed by DAB chromogen development.

• Counterstaining: Counterstain with hematoxylin for nuclear visualization.

• Digital imaging: For quantitative analysis, digital imaging can be performed using systems like the BLISS "Virtual Microscopy" microscope with automated slide scanning capabilities .

How can CC2D1A antibodies be used to investigate protein-protein interactions?

Several techniques have been employed to study CC2D1A interactions with other proteins:

• Co-immunoprecipitation (Co-IP): This has been used to investigate potential interactions between CC2D1A and components of the NFκB pathway. For example, studies examined possible direct interactions between CC2D1A protein and the p65 and p50 subunits of the NFκB complex using co-immunoprecipitation of endogenous proteins in lymphoblastoid cells .

• Proximity ligation assays: These provide higher sensitivity for detecting protein-protein interactions in situ.

• Pull-down assays: His-tagged CC2D1A can be used in pull-down experiments to identify binding partners.

• Yeast two-hybrid screening: This approach has identified novel CC2D1A interaction partners involved in neuronal signaling pathways.

When designing interaction experiments, consider that CC2D1A is distributed throughout the cytoplasm with no nuclear signal detected, suggesting cytoplasmic interactions should be the primary focus .

How can I investigate CC2D1A's role in neuronal development and synaptic plasticity?

To study CC2D1A's neuronal functions, researchers can employ these methodological approaches:

• Conditional knockout models: Use Cre/loxP recombinase-based strategies to conditionally delete CC2D1A exclusively from excitatory neurons of mouse forebrain, as demonstrated in studies examining its role in hippocampal synaptic plasticity .

• Electrophysiological recordings: Measure long-term potentiation (LTP) in hippocampal slices to assess synaptic plasticity changes. CC2D1A cKO mice displayed a deficit in the maintenance of LTP in the CA1 region of hippocampal slices .

• Behavioral testing: Assess cognitive function through:

  • Object location memory tests

  • Anxiety-like behavior evaluations

  • Spatial memory tasks

• Neuronal morphology analysis: Examine dendritic complexity using Golgi staining or fluorescent labeling. CC2D1A deletion resulted in decreased complexity of apical and basal dendritic arbors of CA1 pyramidal neurons .

• Molecular pathway analysis: Investigate Rac1 activity, as enhanced basal Rac1 activity has been observed following CC2D1A deletion, mediated by reduced SUMO-specific protease expression and increased Rac1 SUMOylation .

What approaches can help elucidate CC2D1A's function in ciliary biology and heterotaxy?

Recent studies have revealed CC2D1A's critical role in ciliogenesis. To investigate this function:

• Expression analysis: Examine CC2D1A expression in ciliated tissues using immunofluorescence with validated antibodies. Expression has been confirmed in the left-right organizer, epidermis, pronephric duct, nephrostomes, and ventricular zone of the brain .

• Animal models: Utilize models such as Xenopus, where loss of cc2d1a leads to cardiac heterotaxy, cystic kidneys, and abnormal CSF circulation via defective ciliogenesis .

• Patient-derived fibroblasts: Analyze ciliogenesis in fibroblasts from patients with CC2D1A mutations. These have confirmed defective ciliogenesis in human cells .

• Ciliary function assays: Measure ciliary beat frequency and fluid flow in relevant tissues.

• High-resolution imaging: Use techniques like super-resolution microscopy to examine ciliary ultrastructure in CC2D1A-deficient cells.

How can I investigate CC2D1A's role in immune signaling pathways?

CC2D1A has been identified as a regulator of RIG-I-like receptor (RLR) pathways in antiviral immunity:

• Reporter assays: Overexpression of CC2D1A has been shown to cause significant activation of multiple immune-related transcription factors, with NF-κB showing the strongest response (25.2- ± 7.9-fold increase compared to control cells) .

• Interaction studies: Investigate how TAPE/CC2D1A functionally and physically interacts with RIG-I, MDA5, and IPS-1 to activate the IFN-β promoter .

• Knockdown experiments: TAPE knockdown impairs IFN-β activation induced by RLRs but not IPS-1 .

• Viral challenge models: Assess cytokine production and antiviral responses during RNA virus infection in CC2D1A knockdown or deficient cells .

• Signaling pathway analysis: Examine the activation of downstream effectors in the RLR-IPS-1 pathways in the presence and absence of CC2D1A.

What are common issues when working with CC2D1A antibodies and how can they be resolved?

Researchers may encounter several challenges when using CC2D1A antibodies:

How can I distinguish between CC2D1A and its paralog CC2D1B in my experiments?

CC2D1A has a paralog, CC2D1B, which shares structural similarities and may confound experimental results:

• Antibody selection: Choose antibodies raised against regions with minimal sequence homology between CC2D1A and CC2D1B.

• Western blot differentiation: CC2D1A and CC2D1B have different molecular weights (104 kDa vs. 93 kDa), allowing differentiation on Western blots.

• siRNA/shRNA validation: Confirm specificity of knockdown approaches by demonstrating selective reduction of target protein without affecting the paralog.

• Expression pattern analysis: CC2D1A and CC2D1B show distinct tissue expression patterns that can help differentiate their functions.

• Isoform-specific PCR: Design primers that specifically amplify CC2D1A or CC2D1B transcripts for expression analysis.

How can CC2D1A antibodies be used to study the molecular mechanisms of intellectual disability?

To investigate CC2D1A's role in intellectual disability, researchers can:

• Patient-derived cells: Use CC2D1A antibodies to characterize protein expression, localization, and interactions in neurons derived from patient iPSCs.

• Synaptic protein analysis: Examine how CC2D1A mutations affect the expression and localization of synaptic proteins using co-immunostaining approaches.

• Signaling cascade investigation: As CC2D1A is a putative signal transducer participating in positive regulation of I-κB kinase/NFκB cascade , analyze how mutations disrupt this signaling pathway.

• Structural studies: Combine antibodies with structural biology techniques to understand how specific mutations (e.g., c.575C>T, c.1517A>G, c.1595C>T) impact protein conformation and interaction capabilities .

• Molecular dynamics validation: Use antibodies to validate predictions from molecular dynamics simulations regarding how missense mutations affect protein stability and interactions .

What are promising approaches to study CC2D1A's role in ciliopathies?

Building on recent discoveries linking CC2D1A to ciliary function:

• Advanced imaging: Combine CC2D1A antibodies with super-resolution microscopy to localize the protein within ciliary substructures.

• Developmental timing analysis: Track CC2D1A expression during embryonic development in ciliated tissues to determine critical windows for intervention.

• Rescue experiments: Test whether wild-type CC2D1A expression can rescue ciliary defects in patient-derived cells or animal models.

• Protein interaction networks: Identify ciliary proteins that interact with CC2D1A using proximity labeling approaches combined with immunoprecipitation.

• Cerebrospinal fluid flow analysis: Investigate how CC2D1A mutations affect CSF circulation, particularly in the midbrain region where abnormal local CSF flow has been observed in mutant models .

How might CC2D1A antibodies contribute to potential therapeutic strategies?

Antibody-based approaches could inform therapeutic development:

• Target identification: Use CC2D1A antibodies to identify druggable sites within CC2D1A or its interaction partners.

• Mutation-specific effects: Characterize how different mutations affect CC2D1A localization, providing insights for mutation-specific therapeutic approaches.

• Rac1 pathway intervention: Given that partial blockade of Rac1 activity rescued impairments in LTP and object location memory performance in CC2D1A cKO mice , antibodies could help validate Rac1 pathway modulators as potential therapeutics.

• Biomarker development: CC2D1A antibodies might help identify downstream biomarkers of pathway dysregulation for monitoring treatment efficacy.

• Precision medicine approaches: Link specific mutations to clinical phenotypes to inform tailored intervention strategies .