CCAR2 Antibody

Introduction to CCAR2 and CCAR2 Antibodies

CCAR2, previously known as deleted in breast cancer 1 (DBC1) or KIAA1967, is a multifaceted nuclear protein involved in various cellular processes including DNA damage response, apoptosis, metabolism, chromatin structure, and transcription regulation . CCAR2 antibodies are immunological reagents specifically designed to detect and bind to the CCAR2 protein, enabling researchers to investigate its expression, localization, and interactions within cells and tissues .

The CCAR2 protein plays critical roles in maintaining cellular homeostasis through its involvement in multiple signaling pathways. In humans, the canonical CCAR2 protein has a reported length of 923 amino acid residues and a theoretical molecular mass of 102.9 kDa, although it typically appears at approximately 130 kDa in western blot analyses due to post-translational modifications .

Structure and Characteristics of CCAR2 Protein

CCAR2 is primarily localized in the nucleus and cytoplasm of cells. Its subcellular distribution is particularly important for its diverse functions in regulating transcriptional processes . The protein is an important component of the DBIRD complex, which acts at the interface between core mRNP particles and RNA polymerase II (RNAPII), integrating transcript elongation with the regulation of alternative splicing .

The CCAR2 protein affects local transcript elongation rates and alternative splicing of a large set of exons embedded in (A+T)-rich DNA regions. This function highlights its significance in gene expression regulation at the transcriptional and post-transcriptional levels .

Functions of CCAR2 in Cellular Processes

CCAR2 regulates diverse cellular functions including transcription, mRNA splicing, metabolism, and circadian cycle . One of its most studied functions is its role in the DNA damage response pathway. Upon DNA lesions, CCAR2 is phosphorylated by the apical kinases ATM/ATR, enhancing its binding to SIRT1, leading to SIRT1 inhibition, p53 acetylation, and p53-dependent apoptosis .

Recent research has demonstrated that CCAR2 governs mitotic events, including proper chromosome segregation and cytokinetic division, to maintain chromosomal stability . CCAR2-deficient cells possess multilobulated nuclei, suggesting a defect in cell division. In particular, the duration of the mitotic phase is perturbed due to premature loss of cohesion with the centromere and inactivation of the spindle assembly checkpoint during prometaphase and metaphase .

Additionally, CCAR2 is involved in:

1. Regulating Chk2 activity towards KAP1 phosphorylation in DNA damage response

2. Promoting the stability of transcription factors RFX1 and CREB1

3. Functioning as a coregulator of various transcription factors and a critical regulator of numerous epigenetic modifiers

4. Inhibiting the deacetylase activity of SIRT1 through direct interaction

Types and Properties of CCAR2 Antibodies

CCAR2 antibodies are available in various formats, including monoclonal and polyclonal antibodies, with different host species and conjugation options to suit diverse experimental requirements.

Table 2: Types and Properties of CCAR2 Antibodies

Monoclonal CCAR2 Antibodies

Monoclonal antibodies against CCAR2 offer high specificity and consistency between batches. These antibodies are typically developed using CCAR2 fusion proteins as immunogens and are purified using protein A affinity chromatography . Mouse-derived monoclonal antibodies (such as 66497-1-Ig) demonstrate reactivity with human and pig samples, making them suitable for comparative studies across these species .

Polyclonal CCAR2 Antibodies

Polyclonal antibodies (such as 22638-1-AP) recognize multiple epitopes on the CCAR2 protein, potentially providing enhanced sensitivity for detecting the target protein under various experimental conditions. These antibodies typically show broader species reactivity, including human, mouse, and rat samples .

Conjugated CCAR2 Antibodies

For direct fluorescence detection applications, conjugated antibodies such as CoraLite®594-labeled CCAR2 antibodies (CL594-66497) are available. These fluorescently tagged antibodies eliminate the need for secondary antibody incubation, streamlining immunofluorescence experiments. The CoraLite®594 dye has excitation/emission maxima wavelengths of 588 nm/604 nm .

Applications of CCAR2 Antibodies in Research

CCAR2 antibodies are valuable tools for investigating the expression, localization, and interactions of CCAR2 protein across various experimental contexts. The principal applications include:

Western Blotting (WB)

Western blotting is one of the most common applications for CCAR2 antibodies, enabling detection of CCAR2 protein in cell and tissue lysates. In WB applications, CCAR2 typically appears as a band at approximately 130 kDa . The recommended dilution ranges for CCAR2 antibodies in WB applications vary from 1:2000 to 1:16000, depending on the specific antibody and sample type .

Immunohistochemistry (IHC)

CCAR2 antibodies are effectively used for examining CCAR2 expression patterns in tissue sections. In normal skin, CCAR2 is expressed in both the interfollicular epidermis and the hair follicle, while in cutaneous squamous cell carcinoma (SCC), CCAR2 shows distinct expression patterns that can be detected using IHC . The recommended dilution for IHC applications typically ranges from 1:250 to 1:32000 .

Immunofluorescence (IF)/Immunocytochemistry (ICC)

For cellular localization studies, CCAR2 antibodies are employed in IF/ICC applications. These techniques reveal that CCAR2 predominantly localizes to the nucleus but can also be found in the cytoplasm of cells . The recommended dilution ranges for IF/ICC applications vary from 1:20 to 1:1600 .

Additional Applications

CCAR2 antibodies have been validated for several other specialized applications:

• Flow Cytometry (FC): For quantitative analysis of CCAR2 expression at the single-cell level

• Immunoprecipitation (IP): For isolating CCAR2 protein complexes

• Co-Immunoprecipitation (CoIP): For investigating protein-protein interactions involving CCAR2

• RNA Immunoprecipitation (RIP): For studying RNA-protein interactions with CCAR2

• ELISA: For quantitative measurement of CCAR2 protein levels

Table 3: Recommended Dilutions for Different Applications

Role of CCAR2 in Cancer and Disease

CCAR2 has emerged as a significant player in cancer biology, with complex and sometimes contradictory roles depending on the cellular context and cancer type.

CCAR2 Expression in Cancer

CCAR2 expression has been reported to gradually increase with the progression of gastric carcinoma . High expression of CCAR2 correlates with poor outcomes in many human tumor types, including squamous cell carcinoma (SCC) . Studies have shown that CCAR2 protein levels are significantly elevated in SCC cell lines compared to normal primary keratinocytes due to increased protein stability .

In normal skin, CCAR2 is expressed in both the interfollicular epidermis and hair follicles. Similar expression patterns have been observed in cutaneous SCC, but with notably higher expression levels compared to normal tissues .

Dual Role in Tumorigenesis

Interestingly, CCAR2 appears to play a dual role in cancer progression:

1. Tumor Suppressor Function: Based on its ability to stimulate apoptosis by activating and stabilizing p53, CCAR2 was initially considered to be a tumor suppressor. Loss of Ccar2 in mouse models results in an increased tumor burden, further supporting its tumor-suppressive role .

2. Tumor-Promoting Function: Paradoxically, CCAR2 also functions as a tumor-promoting coregulator by activating oncogenic transcription factors and regulating the enzymatic activity of epigenetic modifiers, indicating that CCAR2 may play a dual role in cancer progression .

The tumor suppressor function of CCAR2 appears to be dependent on p53, a protein that is inactivated in the vast majority of SCC tumors. In p53-null tumors, CCAR2 seems to promote tumorigenesis instead .

Recent Research Findings on CCAR2

Recent studies have revealed several novel functions and mechanisms of CCAR2 in cellular processes:

CCAR2 in Mitotic Regulation

CCAR2 has been identified as a regulator of mitotic progression through spatiotemporal control of Aurora B activity. CCAR2-deficient cells show disturbances in mitotic progression resulting from premature loss of cohesion with the centromere and inactivation of the spindle assembly checkpoint during prometaphase and metaphase. This leads to the formation of lagging chromosomes during anaphase and ultimately activates the abscission checkpoint to halt cytokinesis .

CCAR2 in DNA Damage Response

CCAR2 plays a critical role in the DNA damage response pathway by regulating Chk2 activity towards KAP1. Upon DNA damage, CCAR2 influences Chk2 homodimerization, which is required for its autophosphorylation and activity. In CCAR2-deficient cells, Chk2 homodimerization is impaired, leading to decreased phosphorylation of KAP1 and defective repair of heterochromatic DNA lesions .

CCAR2 in Transcriptional Regulation

CCAR2 has been identified as a master regulator of transcriptional processes through its interactions with various transcription factors and epigenetic modifiers. It forms tissue-specific complexes with proteins such as CECR2 and LUZP1, affecting gene expression in a context-dependent manner .

Antigen Retrieval for IHC Applications

For optimal results in immunohistochemistry applications, antigen retrieval methods are crucial. For CCAR2 antibodies, manufacturers often suggest antigen retrieval with TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative option .

Validation Methods

CCAR2 antibodies undergo rigorous validation through various methods:

• Detection of endogenous CCAR2 in different cell and tissue types

• siRNA-mediated knockdown experiments to confirm specificity

• Comparison with other independent antibodies targeting different epitopes of CCAR2

• Testing reactivity across multiple species

Optimizing Experimental Conditions

For best results with CCAR2 antibodies, researchers should:

1. Titrate the antibody in each testing system to determine optimal concentrations

2. Include appropriate positive and negative controls

3. Consider sample-dependent factors that may affect antibody performance

4. Follow manufacturer-specific protocols for each application