CXorf21 Antibody

What is CXorf21 and why is it significant for autoimmune research?

CXorf21 (Chromosome X Open Reading Frame 21) is an X-linked gene that escapes X-inactivation and is significantly associated with SLE and pSS pathogenesis. The protein is predominantly expressed in immune cells, particularly antigen-presenting cells like monocytes and B cells, with higher expression levels in females compared to males . CXorf21's significance stems from several key characteristics:

• It represents a direct molecular link to female-biased autoimmune diseases

• It escapes X-inactivation, resulting in higher expression in female cells

• It interacts with SLC15a4, another autoimmune risk factor, affecting lysosomal pH

• It participates in TLR7-mediated immune responses, which are central to autoantibody production

• Expression increases after TLR7 and NOD1 stimulation, particularly in female cells

Recent studies have shown that expression levels of CXorf21 are elevated in both male and female SLE patients, with even higher expression in individuals with X-chromosome aneuploidies (47,XXX females and 47,XXY males) .

What techniques can be used to detect CXorf21 in research samples?

CXorf21 can be detected using multiple complementary laboratory techniques:

• Western Blot Analysis: Using anti-CXorf21 antibodies (such as those targeting AA 165-215) with appropriate secondary detection systems. CXorf21 appears at approximately 34 kDa, and bands can be quantified using densitometry software like ImageJ .

• Quantitative PCR: Validated primers targeting exon junctions (e.g., PrimePCR™ probes targeting exon 1 and 2) with reference genes like GAPDH for normalization .

• Immunohistochemistry: Polyclonal antibodies can be used at 1:200-400 dilution on paraffin-embedded sections .

• Flow Cytometry: Available antibodies conjugated to various fluorophores (FITC, AbBy Fluor® 350, 488, 647, 680) enable detection in specific immune cell populations .

• Immunofluorescence: Both unconjugated and fluorophore-conjugated antibodies can visualize subcellular localization .

For comprehensive analysis, researchers often combine these methods to validate findings across different experimental platforms.

How does CXorf21 expression differ between males and females in immune cells?

CXorf21 shows distinct sex-based expression patterns in immune cells:

Female primary monocytes and B cells consistently demonstrate significantly higher basal CXorf21 gene and protein expression compared to matched male cells . This expression difference is further amplified following stimulation with TLR7 (Imiquimod) and NOD1 (iE-DAP) agonists, resulting in enhanced cytokine production (IL-6 and TNF-alpha) in female cells . The sexually dimorphic expression pattern correlates with the strong female bias observed in SLE and pSS, suggesting CXorf21 may be a fundamental contributor to sex differences in autoimmune pathogenesis .

What are the best practices for validating CXorf21 antibody specificity?

Ensuring antibody specificity is crucial for reliable research. For CXorf21 antibodies, implement these validation steps:

• Positive and negative controls: Use cell types known to express CXorf21 (monocytes, B cells) as positive controls and cells with minimal expression (or CRISPR-Cas9 knockout cells) as negative controls .

• Knockdown validation: Use CRISPR-Cas9 targeting exon 1 of CXorf21 (chr.X:30560017-30560036) to generate knockdown models for antibody validation .

• Western blot verification: Confirm the expected molecular weight (~34 kDa) with appropriate ladders .

• Cross-reactivity assessment: Verify species reactivity claims (human, mouse, rat) with appropriate samples .

• Alternative antibodies: Compare results using antibodies targeting different epitopes (AA 165-215 vs. AA 1-301) .

• Peptide competition: For immunohistochemistry applications, perform peptide competition assays using the KLH conjugated synthetic peptide derived from human CXorf21 .

Proper validation not only ensures specificity but also enables reliable interpretation of sex-based differences in CXorf21 expression.

How should researchers design CXorf21 knockout or knockdown experiments?

For effective CXorf21 loss-of-function studies:

• CRISPR-Cas9 approach:

  • Design gRNA targeting exon 1 of CXorf21 (chr.X:30560017-30560036)

  • Form Cas9 RNP nuclease and CXorf21 gRNA complexes using CRISPRMAX Lipofectamine reagent

  • Include appropriate controls (control gRNA, GFP transfection control)

  • Validate knockout efficiency using Genomic Detection Cleavage kit

  • Confirm protein reduction by Western blot with CXorf21 antibodies

• Experimental timeline:

  • Transfect for 48 hours

  • Change media after 8 hours and allow incubation for additional 40 hours

  • After transfection, allow cells to recover overnight before TLR7 stimulation

• Functional readouts:

  • Measure cytokine production (IL-6, TNF-alpha) after TLR7 stimulation

  • Assess lysosomal pH using pHrodo™ assays

  • Compare responses between male and female cells

Researchers should note that CXorf21 knockdown has demonstrated sex-specific effects, with significant impact on female cells but minimal effect in male cells, highlighting the importance of including both sexes in experimental design .

What protocols are recommended for measuring lysosomal pH in relation to CXorf21 function?

To assess CXorf21's role in lysosomal pH regulation:

• pHrodo™ lysosomal pH experiments:

  • Isolate primary classical CD14++/CD16− monocytes from healthy donors of both sexes

  • Allow cells to adhere to culture plates for further purification

  • Perform CXorf21 knockdown using CRISPR-Cas9 (as described above)

  • Load cells with pH-sensitive pHrodo™ dye according to manufacturer's protocol

  • Measure fluorescence intensity changes that reflect lysosomal pH

  • Compare wildtype, CXorf21 knockdown, and control knockdown cells

• Data interpretation:

  • Knockdown of CXorf21 in female monocytes increases lysosomal pH (shifting from acidic to more alkaline)

  • This mimics what happens with SLC15a4 deficiency

  • The pH change disrupts (auto)antigen processing in female samples

  • Male samples typically show less dramatic changes in pH with CXorf21 knockdown

This approach has revealed that CXorf21 appears to maintain the lysosomal pH gradient necessary for monocyte and B cell immune response, providing mechanistic insight into how its sexually dimorphic expression affects autoimmune pathogenesis .

How can researchers effectively detect CXorf21 by Western blotting?

For optimal Western blot detection of CXorf21:

• Sample preparation:

  • Use fresh samples when possible

  • Include protease inhibitors in lysis buffers

  • Prepare SDS-PAGE according to Laemmli method

  • Use pre-cast 4–20% gradient gels for optimal resolution

• Electrophoresis and transfer:

  • Transfer proteins to nitrocellulose membranes using Trans-Blot Turbo transfer system

  • Verify transfer efficiency with Ponceau staining

• Antibody incubation:

  • Block membranes appropriately (usually 5% non-fat milk or BSA)

  • Apply anti-CXorf21 antibody at recommended dilution (1:300-5000)

  • For biotin-conjugated antibodies, use appropriate streptavidin detection systems

  • Include anti-actin antibody as loading control

• Detection and quantification:

  • Use alkaline phosphatase/nitro blue tetrazolium/5-bromo-5-chloro-3-indolyl phosphate system for detection

  • Quantify protein bands using densitometry (ImageJ)

  • Express results relative to housekeeping proteins and compare between experimental groups

This approach has been successfully used to demonstrate increased CXorf21 expression in LCLs from SLE patients and individuals with X-chromosome aneuploidies, as well as to validate knockdown efficiency .

How can researchers investigate CXorf21's interaction with SLC15a4?

To explore the relationship between CXorf21 and SLC15a4:

• Co-immunoprecipitation studies:

  • Prepare cell lysates from relevant immune cells (monocytes, B cells)

  • Immunoprecipitate with anti-CXorf21 antibody

  • Analyze precipitated proteins by Western blot with anti-SLC15a4 antibody

  • Perform reciprocal Co-IP (precipitate with SLC15a4, blot for CXorf21)

  • Include appropriate controls (IgG control, knockdown controls)

• Proximity ligation assay (PLA):

  • Fix and permeabilize cells on slides

  • Incubate with primary antibodies against CXorf21 and SLC15a4 (from different species)

  • Apply PLA probes and perform ligation and amplification

  • Visualize interaction signals by fluorescence microscopy

  • Quantify PLA signals to measure interaction strength

• Functional studies:

  • Generate single and double knockdowns of CXorf21 and SLC15a4

  • Assess lysosomal pH using pHrodo™ assays

  • Measure TLR7-stimulated cytokine production

  • Analyze epistatic relationships between these proteins

Research has shown that CXorf21 interacts with SLC15a4, a lysosomal proton-oligopeptide co-transporter essential for endolysosomal antigen processing and TLR7-mediated cytokine and antibody production in dendritic cells and B cells .

How can CXorf21 antibodies be used to study its role in TLR7 signaling pathways?

To investigate CXorf21's involvement in TLR7 signaling:

• Activation studies:

  • Stimulate cells with TLR7 agonist (Imiquimod) and NOD1 agonist (iE-DAP)

  • Measure changes in CXorf21 expression by Western blot and qPCR

  • Assess IFN-alpha and NFkappaB expression changes

  • Quantify IL-6 and TNF-alpha cytokine production

  • Compare responses between male and female cells

• Knockdown impact assessment:

  • Generate CXorf21 knockdown using CRISPR-Cas9 (as described previously)

  • Stimulate with TLR7 agonists

  • Measure cytokine responses in wildtype vs. knockdown cells

  • Compare effects in male vs. female samples

• Signaling pathway analysis:

  • Prepare protein lysates at different time points after TLR7 stimulation

  • Use Western blotting with phospho-specific antibodies against key signaling molecules

  • Compare signaling kinetics between wildtype and CXorf21-deficient cells

  • Analyze differences between male and female cells

Research has demonstrated that TLR7 activation increases CXorf21 expression, creating a potential positive feedback loop. CXorf21 knockdown abrogates cytokine response in female samples but has minimal effect in male subjects, highlighting its sex-specific role in TLR7 signaling .

What approaches can be used to study CXorf21 in patients with X-chromosome aneuploidies?

For investigating CXorf21 in X-chromosome aneuploidies:

• Sample collection:

  • Recruit subjects with various karyotypes:

    • 46,XX females (normal)

    • 46,XY males (normal)

    • 47,XXX females

    • 47,XXY males (Klinefelter syndrome)

  • Include both SLE-affected and unaffected individuals within each karyotype group

  • Collect PBMCs and/or establish lymphoblastoid cell lines (LCLs)

• Expression analysis:

  • Quantify CXorf21 protein levels by Western blot using anti-CXorf21 antibodies

  • Measure CXorf21 mRNA expression by qPCR

  • Compare expression across different karyotypes and disease status

• Functional studies:

  • Assess TLR7-stimulated cytokine production

  • Measure lysosomal pH

  • Correlate with CXorf21 expression levels

Research has shown that in LCLs from SLE-affected subjects, CXorf21 expression levels were increased in 47,XXX/46,XX women (3.8-fold) and 47,XXY/46,XY men (2.3-fold) compared to healthy male control LCLs. Western blot analysis confirmed increased CXorf21 levels in 46,XY and 47,XXY men with SLE . These findings support the hypothesis that X-chromosome gene dosage contributes to SLE risk.

How can researchers investigate the impact of sex hormones on CXorf21 expression?

To explore hormonal influences on CXorf21 expression:

• In vitro hormone treatment:

  • Isolate primary monocytes and B cells from healthy male and female donors

  • Treat cells with physiologically relevant concentrations of:

    • 17β-estradiol (E2)

    • Testosterone

    • Dihydrotestosterone (DHT)

    • Progesterone

  • Include vehicle controls and positive controls (hormone-responsive genes)

  • Treat for various time points (6, 12, 24, 48 hours)

• Expression analysis:

  • Measure CXorf21 protein levels by Western blot with anti-CXorf21 antibodies

  • Quantify CXorf21 mRNA by qPCR

  • Normalize to appropriate housekeeping genes/proteins

  • Compare hormone effects between male and female cells

• Functional assessments:

  • Following hormone treatment, stimulate cells with TLR7 agonists

  • Measure cytokine production

  • Assess lysosomal pH

  • Correlate functional changes with CXorf21 expression

This approach can help determine whether sex hormones directly influence CXorf21 expression, potentially contributing to the sex bias in autoimmune diseases beyond the chromosomal dosage effect.

What are common issues encountered when using CXorf21 antibodies and how can they be resolved?

When working with CXorf21 antibodies, researchers may encounter these challenges:

• Low signal intensity:

  • Increase antibody concentration (try 1:300 instead of 1:5000)

  • Extend incubation time (overnight at 4°C)

  • Use signal amplification (biotin-streptavidin systems)

  • Enrich for cell types with higher CXorf21 expression (monocytes, B cells)

  • Load more protein (50-100 μg total protein)

• High background:

  • Optimize blocking conditions (try different blocking agents)

  • Increase washing steps (at least 3×10 minutes)

  • Reduce antibody concentration

  • Use freshly prepared buffers

  • Test different detection systems

• Inconsistent sex-based differences:

  • Ensure sufficient sample size from both sexes

  • Standardize cell isolation procedures

  • Control for age and other demographics

  • Process samples simultaneously

  • Include appropriate positive controls

• Cross-reactivity issues:

  • Validate with CXorf21 knockdown samples

  • Use antibodies targeting different epitopes

  • Perform peptide competition assays

  • Verify species specificity

Carefully optimizing these parameters will help ensure reliable and reproducible results when studying CXorf21 expression patterns.

How should researchers interpret changes in CXorf21 expression following TLR7 stimulation?

When analyzing TLR7-induced changes in CXorf21 expression:

• Expected patterns:

  • CXorf21 expression increases following TLR7 activation with Imiquimod

  • Female cells typically show more robust increases than male cells

  • Expression changes should correlate with cytokine production (IL-6, TNF-alpha)

  • IFN-alpha and NFkappaB expression also increases alongside CXorf21

• Interpretation framework:

  Response Pattern	Likely Interpretation	Follow-up Investigation
  ↑ CXorf21, ↑ cytokines in females	Normal/expected response	Mechanism linking CXorf21 to cytokine production
  ↑ CXorf21, ↓ cytokines	Potential downstream signaling defect	Examine signaling intermediates
  No CXorf21 change, ↑ cytokines	Alternative pathway activation	Investigate TLR7-independent mechanisms
  Male/female response equivalent	Experimental conditions may affect dimorphism	Titrate stimulus, check cell purity

• Validation approaches:

  • Confirm with both protein (Western blot) and mRNA (qPCR) measurements

  • Include time-course analysis (2, 6, 12, 24 hours post-stimulation)

  • Test dose-response relationship with TLR7 agonist

  • Include CXorf21 knockdown controls

Research has demonstrated that CXorf21 is involved in a sex-dependent dimorphic response to activation through TLR7 and NOD1, with female cells showing exaggerated responses compared to male cells .

How can researchers determine if CXorf21 antibody detects both alleles in female cells?

To assess biallelic detection in female cells:

• SNP-based allele-specific expression:

  • Identify female subjects heterozygous for coding SNPs in CXorf21

  • Extract DNA and RNA from monocytes or B cells

  • Genotype DNA to confirm heterozygosity

  • Perform allele-specific qPCR on cDNA

  • Compare expression ratios to determine escape from X-inactivation

• Immunofluorescence with X-chromosome markers:

  • Perform dual immunofluorescence for CXorf21 and X-inactive specific transcript (XIST)

  • Use confocal microscopy to visualize expression patterns

  • Quantify signal intensity in relation to XIST-marked inactive X chromosome

• Single-cell analysis:

  • Perform single-cell RNA-seq on female immune cells

  • Analyze allele-specific expression patterns

  • Validate protein expression in identified cell populations using CXorf21 antibodies

What standards should be applied when comparing CXorf21 expression between different experimental systems?

For reliable cross-system comparisons:

• Standardized quantification:

  • For Western blots: use common reference samples across experiments

  • For qPCR: apply consistent reference genes and analysis methods

  • For flow cytometry: include calibration beads

• Normalization approaches:

  • Always normalize to appropriate housekeeping controls

  • Present data as relative expression (fold-change)

  • Include common positive controls across experiments

• Reporting standards:

  • Clearly state cell type, preparation method, and stimulation conditions

  • Report demographic details of donors (age, sex, ethnicity)

  • Include detailed methods for protein/RNA isolation and quantification

  • Specify antibody clone, concentration, and incubation conditions

• Statistical considerations:

  • Use appropriate statistical tests based on data distribution

  • Account for multiple comparisons

  • Report both statistical significance and effect size

  • Consider biological versus technical replication

Following these standards ensures that findings regarding CXorf21's sex-biased expression and role in autoimmune pathogenesis can be reliably compared across different experimental systems and research groups.