CXorf56 Antibody

What is CXorf56 and why is it important for cancer research?

CXorf56 is a protein encoded by a gene located on human chromosome Xq24 in a region associated with genomic alterations in patients with syndromic intellectual disability. Recent research has revealed CXorf56's critical role in DNA damage repair pathways, particularly in triple-negative breast cancer (TNBC). CXorf56 protein has been found to increase homologous recombination (HR) repair in TNBC cells by interacting with the Ku70 DNA-binding domain, reducing Ku70 recruitment and promoting recruitment of key HR factors like RPA32, BRCA2, and RAD51 to DNA damage sites .

The protein is particularly important for cancer research because:

Which species reactivity options are available for CXorf56 antibodies?

Commercial CXorf56 antibodies are available with reactivity against multiple species, enabling comparative studies across different model organisms. The primary species options include:

• Human CXorf56 antibodies

• Mouse CXorf56 antibodies

• Rat CXorf56 antibodies

• Other species including cow and dog

This diversity allows researchers to conduct cross-species analyses and utilize various animal models for investigating CXorf56 functions.

What are the common applications for CXorf56 antibodies in research?

CXorf56 antibodies can be utilized across multiple experimental applications, each providing different insights into protein expression, localization, and interaction. Common validated applications include:

• Western Blotting (WB): For quantitative detection of CXorf56 protein expression

• Immunofluorescence (IF): For subcellular localization studies

• Immunohistochemistry (IHC): For tissue expression pattern analysis

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement in solution

The selection of application should be guided by the specific experimental questions being addressed.

What is the subcellular localization of CXorf56 protein?

CXorf56 protein demonstrates a complex subcellular distribution pattern. Research has shown that the protein is primarily localized in:

• Cell nucleus: Where it may participate in DNA damage response pathways

• Cytoplasm: Suggesting potential roles in cytoplasmic signaling

• Endoplasmic reticulum (ER): Where it has been shown to interact with STING protein

When conducting immunofluorescence studies, a 34 kDa immunoreactive band can be detected by CXorf56 antibody in the ER fraction, corresponding to areas also immunolabeled with calnexin (an ER marker) .

How does CXorf56 influence DNA repair pathway choice in cancer cells?

CXorf56 plays a sophisticated role in regulating the balance between homologous recombination (HR) and non-homologous end joining (NHEJ) DNA repair pathways. Experimental evidence shows:

• CXorf56 protein directly interacts with Ku70, a key component of the DNA-PK complex essential for NHEJ repair

• Through this interaction, CXorf56 inhibits Ku70-mediated NHEJ repair

• CXorf56 promotes HR by enhancing recruitment of RPA32, BRCA2, and RAD51 to DNA damage sites

• Linear regression analysis demonstrates a negative correlation between NHEJ and HR efficiency in control, CXorf56-knockdown, and CXorf56 re-expression cells

Mechanistically, CXorf56 appears to function as a molecular switch that biases repair pathway choice toward HR, which is generally considered more accurate than NHEJ. This function has significant implications for cancer therapy strategies targeting DNA repair mechanisms.

What methodological approaches can be used to study CXorf56 function in DNA damage response?

Several sophisticated methodological approaches can be employed to investigate CXorf56's role in DNA damage response:

• CRISPR/Cas9-mediated gene editing:

  • For generating CXorf56 knockout or knockdown cell lines

  • Can be combined with rescue experiments using shRNA-immune cDNA to confirm specificity

• Reporter assays:

  • NHEJ reporter assays to measure NHEJ efficiency

  • HR reporter assays to assess HR efficiency

  • MMEJ (microhomology-mediated end joining) reporter assays to evaluate alternative repair pathways

• Focus formation assays:

  • Immunostaining for DNA damage markers (γ-H2AX) and repair proteins (MDC1, 53BP1, Ku70, RPA32, BRCA2, RAD51)

  • Quantification of foci number and intensity following DNA damage induction

• Pulsed-field gel electrophoresis (PFGE):

  • For direct assessment of DNA damage levels following treatment with DNA-damaging agents

• Cell cycle analysis:

  • Flow cytometry to determine whether repair pathway alterations are due to cell cycle effects

How can CXorf56 antibodies be validated for specificity in experimental systems?

Rigorous validation of CXorf56 antibodies is essential for ensuring experimental reliability. A comprehensive validation strategy should include:

• Western blot analysis with positive and negative controls:

  • Positive controls: Cell lines known to express CXorf56

  • Negative controls: CXorf56 knockdown or knockout cell lines

  • Expected molecular weight confirmation (~34 kDa)

• Peptide competition assays:

  • Pre-incubation of antibody with purified CXorf56 peptide should abolish signal

  • Non-specific peptides should not affect antibody binding

• Multiple antibody comparison:

  • Use of different antibodies targeting distinct CXorf56 epitopes

  • Consistent results across different antibodies suggest specificity

• Cross-species reactivity assessment:

  • Comparing detection patterns in human, mouse, and rat samples

  • Evolutionary conservation of epitopes supports specificity

• Genetic manipulation validation:

  • Correlating antibody signal reduction with CXorf56 knockdown efficiency

  • Restoration of signal with CXorf56 re-expression using shRNA-immune cDNA

What is the relationship between CXorf56 expression and PARP inhibitor sensitivity in cancer cells?

The relationship between CXorf56 expression and PARP inhibitor (PARPi) sensitivity represents a significant area of therapeutic potential. Research has demonstrated:

• CXorf56 knockdown significantly increases TNBC cell sensitivity to olaparib (a PARP inhibitor) both in vitro and in vivo

• Mechanistically, this increased sensitivity appears to be due to compromised HR repair when CXorf56 is depleted

• CXorf56 knockdown has an additive or synergistic effect on PARPi response when combined with BRCA1 or ATM deficiencies

• In mouse xenograft models, tumors with CXorf56 knockdown show more pronounced shrinkage in response to olaparib treatment

This evidence suggests that CXorf56 inhibition could be a viable strategy to induce "BRCA mutation-like effects" and expand the application of PARP inhibitors to treat patients with non-BRCA mutant tumors or to overcome PARP inhibitor resistance in BRCA-mutant tumors.

What are the optimal fixation and antigen retrieval methods for CXorf56 immunohistochemistry?

When performing immunohistochemistry (IHC) with CXorf56 antibodies, optimization of fixation and antigen retrieval protocols is crucial for accurate detection:

• Fixation recommendations:

  • 10% neutral-buffered formalin for 24-48 hours at room temperature

  • Avoid overfixation which can mask epitopes

  • For frozen sections, 4% paraformaldehyde for 10-15 minutes provides adequate fixation

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) is generally effective

  • For some epitopes, EDTA buffer (pH 9.0) may provide superior results

  • Optimal retrieval time should be determined empirically (typically 15-30 minutes)

• Blocking conditions:

  • 5-10% normal serum (species matched to secondary antibody)

  • 1% BSA in PBS to reduce background staining

  • Consider adding 0.1-0.3% Triton X-100 for improved antibody penetration

These protocols should be optimized based on the specific CXorf56 antibody being used and the tissue type under investigation.

What controls should be included in experiments using CXorf56 antibodies?

Rigorous experimental design requires appropriate controls to ensure valid interpretation of results. When working with CXorf56 antibodies, researchers should include:

• Positive controls:

  • TNBC cell lines (e.g., MDA-MB-231) which express high levels of CXorf56

  • Tissues known to express CXorf56 (e.g., breast cancer tissues, particularly TNBC)

• Negative controls:

  • Primary antibody omission control

  • Isotype control antibody (matched to CXorf56 primary antibody)

  • CXorf56 knockdown or knockout cell lines

• Specificity controls:

  • Peptide competition/neutralization assay

  • Multiple antibodies against different epitopes of CXorf56

• Expression validation controls:

  • Correlation of protein detection with mRNA expression

  • Western blot confirming expected molecular weight

Including these controls is essential for distinguishing specific CXorf56 signal from background or non-specific binding.

How should CXorf56 antibody dilution be optimized for different applications?

Optimal antibody dilution varies based on application, antibody source, and sample type. A systematic approach to dilution optimization includes:

Optimization should be performed for each new lot of antibody, as variation between lots can significantly impact optimal working dilution.

How can CXorf56 antibodies be used to study its role in cancer progression?

CXorf56 antibodies enable multiple experimental approaches to investigate its role in cancer progression:

What are the challenges in detecting CXorf56 in various subcellular compartments?

Detecting CXorf56 across its multiple subcellular locations presents several technical challenges:

• Nuclear vs. cytoplasmic vs. ER localization:

  • Different fixation protocols may preferentially preserve certain compartments

  • Permeabilization conditions must be optimized to access all compartments

  • Cell fractionation protocols should be validated to ensure clean separation

• Epitope accessibility:

  • CXorf56's interaction with Ku70 or other proteins may mask certain epitopes

  • Conformational changes between compartments may affect antibody binding

  • Consider using antibodies targeting different epitopes for comprehensive detection

• Signal-to-noise optimization:

  • Nuclear staining often has higher background

  • ER staining requires co-localization with established markers (e.g., calnexin)

  • Cytoplasmic signal can be diffuse and difficult to quantify

• Dynamic trafficking:

  • CXorf56 may shuttle between compartments depending on cellular state

  • DNA damage induction may alter subcellular distribution

  • Live-cell imaging approaches may be needed to capture dynamic localization

How can CXorf56 antibodies be used to investigate its role in DNA repair pathway choice?

Investigating CXorf56's role in DNA repair pathway choice requires sophisticated experimental approaches:

• Chromatin immunoprecipitation (ChIP):

  • Assess CXorf56 recruitment to DNA damage sites

  • Compare with recruitment timing of other repair factors

  • Determine whether CXorf56 and Ku70 binding are mutually exclusive

• Immunoprecipitation-mass spectrometry (IP-MS):

  • Identify CXorf56 interactome in response to DNA damage

  • Compare interacting partners before and after damage induction

  • Map protein-protein interaction domains

• Focus formation assays with sequential staining:

  • Track temporal recruitment of CXorf56 and other repair factors

  • Use laser micro-irradiation to create localized DNA damage

  • Quantify focus formation kinetics with and without CXorf56 depletion

• Domain mapping experiments:

  • Generate deletion mutants to identify critical CXorf56 domains

  • Determine which domains interact with Ku70's DNA-binding domain

  • Assess functional consequences of disrupting specific interactions

• Cell cycle-specific analysis:

  • Synchronize cells at different cell cycle phases

  • Evaluate how CXorf56 affects repair pathway choice specifically during S and G2 phases

What cell types and experimental conditions are optimal for studying CXorf56 protein function?

Optimizing experimental systems for CXorf56 research requires careful consideration of cell types and conditions:

• Recommended cell lines:

  • TNBC cell lines (MDA-MB-231, SUM1315) show high CXorf56 expression

  • Non-TNBC breast cancer lines as comparative models

  • Normal breast epithelial cells as baseline controls

• DNA damage induction methods:

  • Ionizing radiation (IR): 2-10 Gy doses induce robust DNA damage response

  • Chemotherapeutics: Cisplatin (interstrand crosslinks) challenges different repair pathways

  • PARP inhibitors: Olaparib treatment creates replication-dependent DNA damage

• Time course considerations:

  • Early response (0.5-2 hours post-damage): Initial recruitment dynamics

  • Intermediate response (4-8 hours): Repair process completion

  • Late response (24+ hours): Resolution and survival outcomes

  • Studies show CXorf56 depletion results in increased γH2AX focus accumulation 8 hours post-IR

• Culture conditions affecting CXorf56 function:

  • Serum starvation may alter repair pathway preferences

  • Cell density impacts replication stress and baseline damage

  • Oxygen levels can modify DNA damage responses via oxidative stress

How might CXorf56 antibodies contribute to understanding its role in neurodevelopment?

While CXorf56's role in cancer has been recently highlighted, its original identification in the context of neurodevelopment warrants further investigation:

• Neurodevelopmental studies:

  • CXorf56 is located in a region where genomic alterations have been reported in patients with syndromic intellectual disability

  • Antibodies could help map expression patterns during neural development

  • Co-localization studies with neuronal markers across developmental timepoints

• X-inactivation research:

  • CXorf56 has been implicated in X-inactivation processes

  • Antibodies could help track CXorf56 distribution during X-inactivation

  • Single-cell immunofluorescence to examine expression variability

• STING pathway connections:

  • CXorf56 (also known as STEEP1) interacts with STING protein in the ER

  • Antibodies could help clarify this interaction in neuronal contexts

  • Investigate whether neuroinflammatory responses involve CXorf56

• Comparative neurobiology:

  • Cross-species antibody reactivity enables evolutionary studies

  • Map conservation of expression patterns across model organisms

How can CXorf56 antibodies be utilized in developing potential cancer therapeutics?

The role of CXorf56 in DNA repair pathways positions it as a potential therapeutic target:

• Target validation studies:

  • Antibodies can confirm target engagement in drug discovery pipelines

  • Pharmacodynamic biomarker development using CXorf56 levels or localization changes

• Companion diagnostic development:

  • IHC assays using validated CXorf56 antibodies could identify patients likely to respond to therapies targeting CXorf56 or related pathways

  • Expression level cutoffs would need to be standardized and validated

• Antibody-drug conjugate potential:

  • If cell-surface expression is confirmed, CXorf56 antibodies could potentially be developed into therapeutic antibodies

  • Internalization studies would be required to evaluate feasibility

• Combination therapy research:

  • Monitoring CXorf56 expression/function during combination treatments

  • Understanding resistance mechanisms through expression changes

  • Studies show CXorf56/BRCA1 or CXorf56/ATM double knockdown enhances sensitivity to PARP inhibitors

What methodological advances might enhance CXorf56 antibody applications?

Emerging technologies offer opportunities to expand CXorf56 antibody applications:

• Super-resolution microscopy:

  • Improved visualization of CXorf56 localization at DNA damage sites

  • Nanoscale co-localization with repair factors

  • 3D reconstruction of repair complexes

• Proximity labeling approaches:

  • CXorf56 fusion with BioID or APEX2 to map proximal interactome

  • Temporal changes in protein neighborhoods during DNA repair

  • Compartment-specific interactome mapping

• Single-cell approaches:

  • Cellular heterogeneity in CXorf56 expression

  • Correlation with repair efficiency at single-cell level

  • Combined protein-RNA detection (CITE-seq adapted for CXorf56)

• In vivo imaging:

  • Development of fluorescently tagged antibody fragments for in vivo tracking

  • Intravital microscopy to observe CXorf56 dynamics in tumor microenvironments

  • Correlative light-electron microscopy for ultrastructural localization

These methodological advances would significantly expand our understanding of CXorf56 biology and its therapeutic potential in cancer and possibly neurological conditions.