ATP6V1C2 Antibody

What is ATP6V1C2 and why is it important in cancer research?

ATP6V1C2 encodes a component of the cytosolic V1 domain of vacuolar adenosine triphosphatase (V-ATPase), a multisubunit enzyme responsible for transporting hydrogen ions and mediating acidification of eukaryotic intracellular organelles. Recent research has revealed its significant role in cancer development and progression, particularly in colon adenocarcinoma (COAD).

What is the cellular distribution and expression pattern of ATP6V1C2?

ATP6V1C2 displays tissue-specific expression patterns that are important for researchers to consider when designing experiments:

ATP6V1C2 is predominantly expressed in the kidney with high expression in renal intercalated cells (IC) , in contrast to its paralog ATP6V1C1 which shows broader tissue distribution. The protein has a molecular mass of approximately 43-44 kDa as confirmed by Western Blot analyses .

Immunohistochemistry studies have shown that ATP6V1C2 is distributed both in the cytoplasm and on the cellular membrane in COAD tissues . This distribution pattern was consistently observed in samples analyzed through The Human Protein Atlas database .

Positive immunohistochemical detection has been reported in multiple tissue types:

• Human testis tissue

• Human heart tissue

• Human kidney tissue

• Human liver tissue

• Human lung tissue

• Human placenta tissue

This tissue distribution information is critical when selecting positive controls for antibody validation experiments.

What are the best experimental approaches for ATP6V1C2 detection in cancer tissue samples?

For optimal detection of ATP6V1C2 in cancer tissue samples, researchers should consider a multi-method approach:

Western Blot (WB):

• Recommended dilution range: 1:500-1:2000

• Expected molecular weight: 44 kDa

• Positive control samples: Human kidney tissue, mouse kidney tissue

• Sample preparation: Protein extraction using SDS-PAGE followed by transfer to PVDF membrane

• Detection strategy: Primary ATP6V1C2 antibody followed by HRP-conjugated secondary antibody and ECL reagent application

• Normalization control: GAPDH expression is recommended as an internal control

Immunohistochemistry (IHC):

• Recommended dilution range: 1:20-1:200 or 1:150

• Antigen retrieval: TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 can be alternatively used

• Visualization: DAB Kit (such as Gentech, catalog number: GK500710) with light hematoxylin counterstaining

• Expected distribution: Both cytoplasmic and membrane staining

These methodological details ensure reliable and reproducible detection of ATP6V1C2 in experimental settings.

How can researchers effectively design ATP6V1C2 knockdown experiments for functional studies?

Based on published research, effective ATP6V1C2 knockdown experiments should follow these methodological guidelines:

• Selection of appropriate cell models:

  • COAD cell lines have been successfully used in previous studies

  • HEK293T cells have shown good transfection efficiency with ATP6V1C2 constructs

• Knockdown validation:

  • Western blot is crucial for confirming effective knockdown, using antibody dilutions of 1:500-1:1000

  • GAPDH should be used as the normalization control

• Functional readouts to assess:

  • Expression of Wnt- and EMT-related genes, as ATP6V1C2 has been shown to promote EMT by activating the Wnt signaling pathway

  • Cell proliferation and growth assays, as ATP6V1C2 knockdown inhibits COAD cell proliferation

  • Pathway activity of CD8 T effector cells to evaluate tumor microenvironment effects

• Bioinformatic analysis approaches:

  • Protein-protein interaction networks can be analyzed using GeneMANIA (http://genemania.org) and STRING (https://string-db.org/cgi/input.pl) databases

  • Differential gene expression analysis between ATP6V1C2-high and -low expression groups is recommended to identify affected pathways

This systematic approach allows for comprehensive functional characterization of ATP6V1C2 in cancer models.

What are the molecular mechanisms through which ATP6V1C2 contributes to cancer progression?

Recent research has elucidated several key mechanisms through which ATP6V1C2 appears to contribute to cancer progression, particularly in colon adenocarcinoma:

• Epithelial-Mesenchymal Transition (EMT) regulation:

  • High expression of ATP6V1C2 is associated with high EMT scores in bioinformatic analyses

  • ATP6V1C2 knockdown results in aberrant expression of EMT-related genes

• Wnt signaling pathway activation:

  • The Wnt signaling pathway is significantly enriched in differentially expressed genes between ATP6V1C2-high and -low expression groups

  • ATP6V1C2 appears to promote EMT by activating the Wnt signaling pathway

• Tumor microenvironment modulation:

  • High expression of ATP6V1C2 can decrease pathway activity of CD8 T effector cells, potentially contributing to immune evasion

• V-ATPase functional roles:

  • As part of the V-ATPase complex, ATP6V1C2 participates in proton transport and acidification of intracellular compartments

  • The C subunit is necessary for the assembly of the catalytic sector of the enzyme and likely has a specific function in its catalytic activity

These mechanistic insights provide potential intervention points for therapeutic strategies targeting ATP6V1C2 in cancer treatment.

What are the key considerations for selecting an appropriate ATP6V1C2 antibody for specific experimental applications?

Selecting the optimal ATP6V1C2 antibody requires careful consideration of several factors:

Antibody type considerations:

Application-specific recommendations:

• For Western Blot:

  • Recommended antibodies: 16274-1-AP (Proteintech) , ab176771 (Abcam) , or OTI8H4 (Boster)

  • Optimal dilutions: 1:500-1:2000 depending on the specific antibody

  • Expected molecular weight: ~44 kDa

• For Immunohistochemistry:

  • Recommended antibodies: 16274-1-AP with 1:20-1:200 dilution or OTI8H4 with 1:150 dilution

  • Antigen retrieval: TE buffer pH 9.0 is preferred, though citrate buffer pH 6.0 is an alternative

• For Immunofluorescence:

  • Several antibodies have been validated for IF applications

  • Tissue-specific expression patterns should inform control selection

Validation status considerations:

• Verify that the antibody has been validated for your species of interest (most are validated for human and mouse samples)

• Check if knockout/knockdown validation has been performed, which provides the strongest evidence of specificity

Proper antibody selection is critical for obtaining reliable and reproducible results in ATP6V1C2 research.

How does ATP6V1C2 relate to renal physiology and pathology?

ATP6V1C2 plays a significant role in renal physiology and has been implicated in renal pathologies:

• Normal physiological function:

  • ATP6V1C2 is predominantly expressed in the kidney, particularly in renal intercalated cells (IC)

  • As a component of V-ATPase, it participates in proton secretion and pH regulation in the kidneys

  • The protein functions as part of the V1 complex of vacuolar(H+)-ATPase (V-ATPase), which is responsible for acidifying and maintaining the pH of intracellular compartments

• Association with distal renal tubular acidosis (dRTA):

  • Whole exome sequencing has identified ATP6V1C2 as a novel candidate gene for distal renal tubular acidosis

  • Like ATP6V0A4 and ATP6V1B1, ATP6V1C2 encodes a subunit of the V-type proton ATPase critical for renal acid-base homeostasis

  • Deleterious recessive mutations in ATP6V1C2 have been identified in dRTA patients who did not have mutations in previously known dRTA genes

• Functional relationship to other V-ATPase subunits:

  • ATP6V1C2 functions alongside other V-ATPase subunits (including those encoded by ATP6V0A4 and ATP6V1B1) in maintaining acid-base balance in the kidney

  • While its paralog ATP6V1C1 is broadly expressed, ATP6V1C2's kidney-specific expression pattern suggests specialized functions in renal physiology

Understanding ATP6V1C2's role in renal physiology provides important context for both basic research and clinical studies targeting V-ATPase function in kidney diseases.

What methodological approaches are recommended for investigating ATP6V1C2 expression patterns across different tissues?

To comprehensively investigate ATP6V1C2 expression patterns across tissues, researchers should employ multiple complementary approaches:

Transcriptomic analysis:

• RNA-seq data from public databases like TCGA and GEO can provide tissue-specific expression profiles

• Single-cell RNA sequencing can identify cell-type specific expression patterns, especially important for heterogeneous tissues like kidney

Immunohistochemistry methodology:

• Recommended protocol:

  • Formalin-fixed, paraffin-embedded (FFPE) tissue sections

  • Antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0

  • Primary antibody incubation with ATP6V1C2 polyclonal antibody at 1:20-1:200 dilution

  • Visualization using DAB Kit

  • Light hematoxylin counterstaining

• Validated positive control tissues include:

  • Human testis, heart, kidney, liver, lung, and placenta tissues

Western blot analysis:

• Tissue lysates from multiple organs should be compared

• Recommended positive controls: human and mouse kidney tissues

• Protein extraction protocol should use SDS-PAGE followed by transfer to PVDF membrane

• Detection with primary ATP6V1C2 antibody (1:500-1:1000) followed by HRP-conjugated secondary antibody

• GAPDH should be used as loading control for normalization

Bioinformatic validation:

• The Human Protein Atlas (https://www.proteinatlas.org/) can be used to confirm and extend wet-lab findings

• Cross-referencing expression patterns with functional data from differential expression studies can provide biological context

This multi-modal approach provides robust validation of tissue-specific expression patterns and cellular localization of ATP6V1C2.

How does ATP6V1C2 function within the broader V-ATPase complex architecture?

ATP6V1C2 serves specific structural and functional roles within the V-ATPase complex:

Structural position within V-ATPase complex:

V-ATPase is composed of two major domains: a cytosolic V1 domain that hydrolyzes ATP and a membrane-integral V0 domain that translocates protons .

The V1 domain architecture includes:

• Three A subunits

• Three B subunits

• Two G subunits

• One each of C, D, E, F, and H subunits

ATP6V1C2 specifically encodes the C2 subunit isoform within this V1 domain .

Functional role within the complex:

• Assembly regulation:

  • The C subunit is necessary for the assembly of the catalytic sector of the enzyme

  • It likely contributes to the structural stability of the assembled complex

• Catalytic activity:

  • ATP6V1C2 is likely to have a specific function in the complex's catalytic activity

  • The V1 domain containing ATP6V1C2 houses the ATP catalytic site

• Isoform-specific functions:

  • While the general V-ATPase functions in acidification of intracellular compartments, the tissue-specific expression of ATP6V1C2 (predominantly in kidney) suggests specialized roles

  • ATP6V1C2 represents one of multiple V1 domain C subunit isoforms created through alternative splicing

This detailed understanding of ATP6V1C2's position and function within the V-ATPase complex provides important context for interpreting experimental results and designing targeted interventions.

What are the most reliable experimental approaches for studying ATP6V1C2's role in the Wnt signaling pathway?

To rigorously investigate ATP6V1C2's role in Wnt signaling, researchers should employ these experimental approaches:

Pathway activity assessment:

• TOP/FOP flash reporter assay:

  • Transfect cells with TOP-flash (containing TCF binding sites) and FOP-flash (containing mutated sites) reporters

  • Compare luciferase activity ratios between ATP6V1C2 knockdown/overexpression and control conditions

  • This directly measures canonical Wnt pathway transcriptional activity

• β-catenin nuclear translocation:

  • Perform subcellular fractionation followed by Western blot or

  • Conduct immunofluorescence to visualize β-catenin localization

  • ATP6V1C2 appears to activate Wnt signaling, so modulation should affect β-catenin nuclear accumulation

Target gene expression analysis:

• RT-qPCR panel of Wnt target genes:

  • Measure expression of established Wnt targets (AXIN2, CCND1, MYC, etc.)

  • Compare expression in ATP6V1C2 knockdown/overexpression vs. control conditions

  • Normalize to appropriate housekeeping genes

• RNA-seq analysis:

  • Perform differential expression analysis between ATP6V1C2-high and -low expressing samples

  • Conduct pathway enrichment analysis to confirm Wnt pathway enrichment as previously observed

  • Use GSEA to assess enrichment of Wnt signature gene sets

Protein interaction studies:

• Co-immunoprecipitation:

  • Investigate physical interactions between ATP6V1C2 and Wnt pathway components

  • Use tagged constructs or endogenous protein pulldown with specific antibodies

• Proximity ligation assay:

  • Visualize protein-protein interactions in situ

  • Particularly useful for detecting transient or context-dependent interactions

Rescue experiments:

• Pathway modulation:

  • Combine ATP6V1C2 knockdown with Wnt activators (e.g., CHIR99021, Wnt3a)

  • Determine if Wnt pathway activation can rescue phenotypes induced by ATP6V1C2 depletion

• EMT marker rescue:

  • Since ATP6V1C2 promotes EMT through Wnt signaling , assess if direct Wnt activation can restore EMT marker expression in ATP6V1C2-depleted cells