C19orf10 Antibody

What is C19orf10 and what are its known biological functions?

C19orf10 is a protein encoded by the Chromosome 19 Open Reading Frame 10 gene in humans. Initially thought to function as an interleukin supporting lymphoid cell proliferation, current understanding suggests this activity is not consistently reproducible, and its precise function remains under investigation . Recent research has demonstrated that C19orf10 may play a critical role in cancer progression, particularly in kidney renal clear cell carcinoma (KIRC) .

The protein has a molecular weight of approximately 18.6 kDa and is involved in ER-Nucleus Signaling pathways . Structurally, it contains 173 amino acids with the sequence: MAAPSGGWNG VGASLWAALL LGAVALRPAE AVSEPTTVAF DVRPGGVVHS FSHNVGPGDK YTCMFTYASQ GGTNEQWQMS LGTSEDHQHF TCTIWRPQGK SYLYFTQFKA EVRGAEIEYA MAYSKAAFER ESDVPLKTEE FEVTKTAVAH RPGAFKAELS KLVIVAKASR TEL .

The protein demonstrates oncogenic properties in certain cancer types. Experimental evidence indicates that C19orf10 overexpression enhances cell viability and proliferation, while its silencing inhibits cancer cell growth and induces apoptosis .

What experimental approaches are recommended for validating C19orf10 antibody specificity?

Validating antibody specificity is crucial for generating reliable research results. For C19orf10 antibodies, the following methodological approaches are recommended:

• Western Blot Validation: Compare protein expression in cell lines known to express C19orf10 at different levels. Research has validated C19orf10 antibody reactivity in multiple cell lines including A549, LNCaP, HeLa, HepG2, Jurkat, K-562, HSC-T6, and NIH/3T3 cells . This approach allows verification of the expected molecular weight (approximately 18.6 kDa).

• Positive and Negative Controls: Include lysates from cells with C19orf10 knockdown (using siRNAs targeting C19orf10) alongside control cells. Research has demonstrated successful C19orf10 silencing with at least 70% reduction in expression levels using specific siRNAs .

• Cross-Reactivity Testing: Test the antibody against recombinant C19orf10 protein. Commercial recombinant human C19orf10/SF20/IL25 proteins expressed in HEK293 cells with >80% purity (as determined by SDS-PAGE and Coomassie blue staining) can serve as positive controls .

• Immunohistochemical Validation: When performing IHC, include both positive tissue samples (such as KIRC tissues) and negative controls to confirm staining specificity .

What are the optimal storage and handling conditions for C19orf10 antibodies?

To maintain antibody integrity and functionality, the following storage and handling procedures are recommended based on manufacturer guidelines:

• Storage Temperature: Store antibodies at -80°C for long-term preservation. This prevents protein degradation and maintains antibody activity .

• Aliquoting Protocol: Thaw antibodies on ice, then divide into single-use aliquots before re-freezing. This minimizes freeze-thaw cycles which can degrade antibody quality .

• Freeze-Thaw Limitations: Limit to 2-3 freeze-thaw cycles to preserve antibody functionality .

• Buffer Conditions: Optimal buffer conditions include 25 mM Tris.HCl (pH 7.3), 100 mM glycine, and 10% glycerol for maintaining protein stability .

• Working Concentration: Typical working concentration is approximately 50 μg/mL, but this may vary depending on the specific application and antibody .

How can C19orf10 be effectively silenced or overexpressed in experimental models?

Modulating C19orf10 expression is critical for investigating its functional roles. Research has established effective methods for both silencing and overexpression:

For C19orf10 Silencing:

• siRNA Transfection: Two individual siRNAs targeting C19orf10 have demonstrated effective silencing (>70% reduction) in cancer cell lines such as ACHN. The silencing efficiency should be verified by both qPCR and Western blotting .

• Experimental Timeline: Optimal functional assays should be performed 48-72 hours post-transfection when protein knockdown reaches maximum efficiency .

• Verification Methods: Both mRNA (RT-qPCR) and protein (Western blotting) quantification should be performed to confirm knockdown efficiency .

For C19orf10 Overexpression:

• Vector Selection: pCMV6-entry-C19orf10 has been successfully employed for overexpression in cell lines with low endogenous C19orf10 expression (e.g., 769-P cells) .

• Control Selection: pCMV6-entry empty vector serves as an appropriate negative control for overexpression experiments .

• Expression Verification: Both qPCR and Western blotting should be performed to validate successful overexpression .

What role does C19orf10 play in cancer progression, particularly in KIRC?

Research has revealed a significant oncogenic role for C19orf10 in kidney renal clear cell carcinoma (KIRC):

• Differential Expression: C19orf10 is consistently overexpressed in KIRC tissues compared to adjacent non-carcinoma tissues, as demonstrated by qPCR analysis of 30 paired clinical samples .

• Correlation with Disease Progression: Immunohistochemical staining of 33 KIRC patient samples revealed increased C19orf10 expression correlating with advancing histological grade. Expression was significantly higher in high-grade (grade 2-3) compared to low-grade (grade 1-2) tumors .

• Functional Impact on Cancer Hallmarks:

  Cancer Hallmark	Effect of C19orf10 Silencing	Effect of C19orf10 Overexpression
  Cell Viability	Significant reduction	Enhanced viability
  Proliferation	Decreased (reduced EdU-positive cells)	Increased (more EdU-positive cells)
  Apoptosis	Enhanced (increased cleaved caspase 3/7)	Reduced
  Migration	Inhibited	Promoted
  Invasion	Suppressed	Enhanced

• Molecular Mechanisms: C19orf10 potentially exerts its oncogenic effects by regulating the PTEN/Akt pathway and ZO-1, though the exact mechanisms require further investigation .

• Clinical Relevance: C19orf10 expression correlates with advanced TNM stage and presence of lymph node and histological metastasis, suggesting its potential as a biomarker for KIRC progression and prognosis .

What experimental methods are recommended for investigating C19orf10 function in cellular models?

Based on published research, the following methodological approaches are recommended for investigating C19orf10 function:

• Cell Viability Assessment: CCK-8 assay has proven effective for measuring how C19orf10 expression levels affect cell viability in cancer cell lines .

• Proliferation Analysis:

  • EdU incorporation assay provides quantitative assessment of DNA synthesis and cell proliferation

  • The percentage of EdU-positive cells should be quantified across multiple fields (n=8) for statistical reliability

• Apoptosis Detection:

  • Cleaved caspase 3/7 detection effectively measures apoptotic activity following C19orf10 modulation

  • Results should be verified across multiple experimental replicates (n=3)

• Migration and Invasion Assays:

  • Transwell assays without Matrigel coating for migration assessment

  • Matrigel-coated Transwell assays for invasion capability

  • Quantification should include at least 5 independent fields for statistical validity

• Expression Analysis in Cell Lines:

  • RT-qPCR for mRNA quantification

  • Western blotting for protein detection

  • Comparison across multiple cell lines (e.g., cancer cell lines vs. normal control cells like HK-2)

What are the challenges in interpreting C19orf10 antibody staining patterns in clinical samples?

When working with C19orf10 antibodies in clinical samples, researchers face several interpretive challenges:

• Heterogeneous Expression: C19orf10 expression varies across different tumor grades in KIRC, requiring careful stratification of samples by histological grade for accurate interpretation .

• Correlation with Clinical Parameters: Analysis should account for potential confounding factors when correlating C19orf10 expression with clinical parameters such as TNM stage or metastatic status .

• Sample Size Limitations: Current validation studies have used relatively small sample sizes (e.g., 33 KIRC cases for IHC analysis), highlighting the need for larger cohorts to strengthen statistical power .

• Technical Considerations:

  • Antibody specificity must be rigorously validated

  • Standardized scoring systems should be employed for semi-quantitative analysis

  • Both cancer and adjacent normal tissues should be examined as internal controls

• Database Validation: Findings from TCGA and CPTAC database analyses should be validated using independent clinical samples to confirm diagnostic and prognostic potential .

How might C19orf10 serve as a diagnostic or prognostic biomarker in cancer?

Research suggests C19orf10 has significant potential as both a diagnostic and prognostic biomarker, particularly in KIRC:

• Diagnostic Application: Differential expression between tumor and normal tissues suggests C19orf10 could serve as an independent diagnostic marker to discriminate KIRC patients .

• Prognostic Value: Correlation with disease-free survival (DFS) indicates potential utility as a prognostic factor to distinguish high-risk from low-risk patients .

• Risk Stratification: C19orf10 expression correlates with advanced clinical progression in KIRC, suggesting utility in patient risk stratification .

• Implementation Considerations:

  • Standardized detection methods are needed

  • Cut-off values for "high" versus "low" expression should be established

  • Multivariate analysis with established clinical parameters is necessary to confirm independent prognostic value

• Validation Requirements: While initial findings are promising, larger clinical cohorts are needed to validate diagnostic and prognostic potential before clinical implementation .

What are the comparative advantages of different experimental models for studying C19orf10 function?

Different experimental models offer distinct advantages for investigating C19orf10 function:

• Cell Line Models:

  Cell Line	C19orf10 Expression	Advantages	Applications
  ACHN	High	Suitable for knockdown studies	Proliferation, migration, invasion assays
  769-P	Low	Ideal for overexpression studies	Gain-of-function experiments
  HK-2	Baseline (normal control)	Provides normal expression reference	Comparative expression analysis
  A549, LNCaP, HeLa, HepG2, Jurkat, K-562	Positive expression	Diverse tissue origins	Cross-tissue validation

• Patient-Derived Samples:

  • Provide clinically relevant expression patterns

  • Allow correlation with patient outcomes

  • Current limitations include small sample sizes

• Animal Models:

  • Required for in vivo validation of in vitro findings

  • Needed to assess physiological relevance of C19orf10 function

  • Currently represents a research gap, as noted in the literature

What molecular mechanisms underlie C19orf10's cellular functions?

While the complete molecular mechanisms of C19orf10 remain under investigation, current research has identified several potential pathways:

• PTEN/Akt Signaling: C19orf10 appears to regulate the PTEN/Akt pathway, which is crucial for cell survival, proliferation, and metabolism .

• ZO-1 Regulation: C19orf10 may influence ZO-1, suggesting potential roles in cell-cell junction integrity and epithelial-mesenchymal transition .

• ER-Nucleus Signaling: C19orf10 is associated with ER-Nucleus signaling pathways, though the specific mechanisms require further elucidation .

• Research Gaps:

  • Precise protein-protein interactions remain largely uncharacterized

  • Downstream transcriptional targets need identification

  • Potential post-translational modifications and their functional impacts require investigation

  • Detailed structural analyses may provide insights into interaction domains