ATP6V1G1 Antibody

What is ATP6V1G1 and what cellular functions does it perform?

ATP6V1G1 (also known as ATP6G, ATP6G1, ATP6J, V-type proton ATPase subunit G 1) is a subunit of the V1 complex of vacuolar H+ ATPase (V-ATPase), a multisubunit enzyme that consists of a peripheral V1 complex that hydrolyzes ATP and a membrane integral V0 complex that translocates protons . This protein plays a crucial role in acidifying and maintaining the pH of intracellular compartments including lysosomes and endosomes . In certain cell types, it is also targeted to the plasma membrane where it acidifies the extracellular environment . ATP6V1G1 is involved in several critical cellular processes including protein sorting, zymogen activation, receptor-mediated endocytosis, and synaptic vesicle proton gradient generation . Under aerobic conditions, it participates in intracellular iron homeostasis by triggering the activity of Fe(2+) prolyl hydroxylase enzymes, leading to HIF1A hydroxylation and subsequent proteasomal degradation .

What are the common applications for ATP6V1G1 antibodies in research?

ATP6V1G1 antibodies are utilized in multiple experimental applications to investigate the expression and function of this protein. Based on available research data, these antibodies have been validated for Western blotting (WB), immunocytochemistry/immunofluorescence (ICC/IF), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA) . For Western blotting, ATP6V1G1 antibodies are typically used at dilutions ranging from 1:1000 to 1:5000 . For immunohistochemistry applications, dilutions between 1:20 and 1:200 are recommended . For immunofluorescence procedures, the suggested dilution range is 1:50 to 1:200 . These applications enable researchers to detect and quantify ATP6V1G1 expression in various experimental contexts, from cell lines to tissue samples.

What control samples should be included when using ATP6V1G1 antibodies?

When using ATP6V1G1 antibodies, it is essential to include appropriate positive and negative controls to validate experimental findings. For positive controls, researchers should consider using cell lines known to express high levels of ATP6V1G1, such as LN229 and T98G glioblastoma cells . These cell lines have been documented to express elevated levels of ATP6V1G1 compared to less aggressive lines like SW1088 . Protein extracts from GBM neurospheres also show high expression of ATP6V1G1 and can serve as positive controls . For negative or low-expression controls, researchers may use extracts from grade I brain tumors, where V1G1 expression has been reported to be undetectable in protein extracts . Additionally, when performing knockdown or overexpression studies, appropriate vector controls should be included to account for non-specific effects. Using β-actin as a loading control for Western blotting is recommended to normalize protein expression across samples .

How should samples be prepared for optimal ATP6V1G1 antibody detection?

For optimal detection of ATP6V1G1 using antibodies, careful sample preparation is crucial. For protein extraction from adherent cells, RIPA lysate containing cocktail protease phosphatase inhibitor and PMSF pyrolysis should be used . Protein concentration should be determined using the BCA assay prior to SDS-PAGE separation . For Western blotting, proteins should be transferred to a PVDF membrane, which is then blocked in TBST solution containing 5% skim milk for 1 hour before incubation with the primary ATP6V1G1 antibody (typically at a concentration of 1:500) overnight at 4°C . For immunofluorescence analysis, PFA fixation followed by Triton X-100 permeabilization has been successfully used with ATP6V1G1 antibodies at a concentration of 4 μg/ml . When studying phosphorylated forms influenced by ATP6V1G1, special phosphoprotein enrichment techniques like the TiO2 method after trypsin digestion may be necessary .

How can ATP6V1G1 antibodies be used to investigate its role in cancer progression?

ATP6V1G1 has been implicated in the progression of various cancers, including glioblastoma (GBM) and hepatocellular carcinoma (HCC) . To investigate its role in cancer progression, researchers can employ ATP6V1G1 antibodies in a multi-faceted approach. Immunohistochemical analysis of patient tumor samples at different stages can reveal correlations between ATP6V1G1 expression and disease progression, as demonstrated in HCC where ATP6V1G1 expression was shown to be elevated in advanced stages . For mechanistic studies, researchers should consider combining ATP6V1G1 antibody detection with functional assays after genetic manipulation (overexpression or knockdown) of ATP6V1G1 in cancer cell lines.

A comprehensive approach would include:

• Western blotting to quantify ATP6V1G1 protein levels in different cancer cell lines and patient samples

• Immunofluorescence to determine subcellular localization in cancer cells

• Co-immunoprecipitation with ATP6V1G1 antibodies to identify interaction partners

• Phosphoproteomic analysis to identify downstream signaling pathways affected by ATP6V1G1 expression

Studies have shown that ATP6V1G1 expression influences the phosphorylation status of several proteins involved in cancer-related pathways, including p-RPS6(Ser235) and p-SQSTM1(Ser272) (upregulated) and p-PDPK1(Ser241) and p-EEF2(Ser57) (downregulated) . These proteins are involved in critical processes such as the mTOR and PI3K/Akt signaling pathways, autophagy, and protein synthesis, all of which contribute to cancer cell growth and survival .

What are the methodological considerations for studying ATP6V1G1 phosphoregulation using specific antibodies?

• Establish stable cell lines with ATP6V1G1 overexpression or knockdown using lentiviral transfection

• Extract total proteins using SDS lysate with protease and phosphatase inhibitors

• Perform protein reduction, alkylation, and trypsin digestion

• Label peptides with TMT6 labeling reagent for quantitative analysis

• Enrich phosphorylated peptides using the TiO2 method

• Conduct LC-MS/MS analysis to identify phosphorylated proteins

• Validate key findings using western blotting with specific phospho-antibodies

For western blot validation, researchers should use antibodies specific to phosphorylated forms of proteins of interest, such as p-RPS6(Ser235), p-SQSTM1(Ser272), p-PDPK1(Ser241), and p-EEF2(Ser57) . It is essential to normalize results against total protein levels and use appropriate loading controls. RT-PCR should be employed to confirm successful genetic manipulation at the transcript level before proceeding with protein analysis .

How can ATP6V1G1 antibodies be used to study its role in cancer stem cells and therapy resistance?

ATP6V1G1 has been found to be highly expressed in cancer stem cell-enriched neurospheres isolated from glioblastoma patients, suggesting its potential role in cancer stemness . To investigate this aspect, researchers can employ ATP6V1G1 antibodies in several experimental approaches:

• Compare ATP6V1G1 expression between cancer stem cell populations and differentiated tumor cells using western blotting and immunofluorescence

• Analyze patient-derived neurospheres and their corresponding differentiated cultures for ATP6V1G1 expression

• Perform functional assays after manipulating ATP6V1G1 expression in cancer stem cells to assess self-renewal, differentiation, and tumorigenicity

Studies have demonstrated that ATP6V1G1 gene and protein expression significantly decrease when GBM neurospheres are differentiated into adherent cell monolayers, indicating its potential role in maintaining stemness . For therapy resistance studies, researchers can investigate the correlation between ATP6V1G1 expression and response to standard treatments. The phosphorylation of SQSTM1, which is regulated by ATP6V1G1, has been shown to be closely associated with cisplatin resistance in tumor cells , suggesting a potential mechanism through which ATP6V1G1 might influence therapy resistance.

What technical challenges might arise when using ATP6V1G1 antibodies in multiplexed immunoassays?

When incorporating ATP6V1G1 antibodies into multiplexed immunoassays, researchers may encounter several technical challenges that require careful consideration:

• Cross-reactivity: ATP6V1G1 has several related isoforms (ATP6V1G2, ATP6V1G3) and researchers must ensure the antibody specifically detects ATP6V1G1 without cross-reacting with these paralogues .

• Antibody compatibility: In multiplexed fluorescence immunoassays, antibodies must be compatible in terms of species origin to avoid cross-reactivity between secondary antibodies. ATP6V1G1 antibodies are commonly available in rabbit polyclonal format , which may limit combinations with other rabbit-derived antibodies.

• Signal interference: When studying phosphorylated proteins regulated by ATP6V1G1, phospho-specific antibodies may have different optimal conditions for antigen retrieval and detection compared to ATP6V1G1 antibodies.

• Epitope accessibility: As ATP6V1G1 functions as part of a multisubunit complex, epitope masking may occur depending on complex formation, potentially affecting antibody binding.

To overcome these challenges, researchers should:

• Perform careful antibody validation using positive and negative controls

• Use antibodies raised in different host species when designing multiplexed assays

• Consider sequential rather than simultaneous staining for challenging combinations

• Employ spectral unmixing techniques to resolve overlapping fluorescence signals

• Validate findings with alternative methods such as mass spectrometry-based approaches

How can ATP6V1G1 antibodies be used to investigate its role in lysosomal function and autophagy?

ATP6V1G1, as a component of V-ATPase, plays a critical role in maintaining the acidic environment of lysosomes necessary for proper degradation of cellular components through autophagy . To investigate this function, researchers can employ ATP6V1G1 antibodies in conjunction with autophagy markers and functional assays:

• Co-localization studies: Use immunofluorescence with ATP6V1G1 antibodies alongside lysosomal markers (LAMP1, LAMP2) and autophagy markers (LC3, SQSTM1/p62) to assess the association of ATP6V1G1 with these structures.

• Autophagic flux assays: Manipulate ATP6V1G1 expression and monitor changes in autophagy markers by western blotting. The relationship between ATP6V1G1 and SQSTM1 phosphorylation is particularly relevant, as SQSTM1 is a key protein in autophagy formation and its phosphorylation status affects autophagy activity .

• Lysosomal function assessment: Use lysosomal pH-sensitive dyes to determine if ATP6V1G1 manipulation affects lysosomal acidification. Previous research has shown that altering ATP6V1G1 expression (either overexpression or silencing) slowed acid kinetics in HeLa cells, suggesting impaired lysosomal function .

• Proteolytic activity assays: Measure the activity of lysosomal enzymes that depend on acidic pH after ATP6V1G1 manipulation to assess functional consequences on lysosomal degradation.

Research has shown that ATP6V1G1 regulates liver lipid metabolism by maintaining normal acidification function of lysosomes, suggesting its potential role in non-alcoholic fatty liver disease . This connection between ATP6V1G1, lysosomal function, and lipid metabolism represents an important area for further investigation.

What approaches can be used to study ATP6V1G1 interactions with other V-ATPase subunits using antibodies?

Studying the interactions between ATP6V1G1 and other V-ATPase subunits is crucial for understanding the assembly and function of the V-ATPase complex. Several antibody-based approaches can be employed:

• Co-immunoprecipitation (Co-IP): Use ATP6V1G1 antibodies to pull down the protein and its interacting partners, followed by western blotting with antibodies against other V-ATPase subunits. This approach can identify direct or indirect interactions within the complex.

• Proximity ligation assay (PLA): This technique can detect protein-protein interactions in situ by generating a fluorescent signal when two proteins are in close proximity. It requires antibodies against ATP6V1G1 and potential interacting partners raised in different species.

• Cross-linking immunoprecipitation: Use chemical cross-linkers to stabilize protein-protein interactions before immunoprecipitation with ATP6V1G1 antibodies, followed by mass spectrometry to identify interacting proteins.

• Immunofluorescence co-localization: Perform multi-color immunofluorescence using ATP6V1G1 antibodies alongside antibodies against other V-ATPase subunits to assess their spatial co-localization in cells.

ATP6V1G1 is known to be part of the peripheral V1 complex that hydrolyzes ATP , and understanding its interactions with other subunits can provide insights into how V-ATPase assembly and activity are regulated in different cellular contexts, particularly in pathological conditions where ATP6V1G1 expression is altered.

How can researchers validate ATP6V1G1 knockdown or overexpression models using antibodies?

Proper validation of ATP6V1G1 genetic manipulation models is essential for ensuring experimental reliability when studying its functions. ATP6V1G1 antibodies play a crucial role in this validation process:

• Western blotting: The primary method to confirm successful manipulation of ATP6V1G1 at the protein level. For knockdown validation, researchers should observe a significant reduction in ATP6V1G1 protein levels compared to control samples. For overexpression studies, increased protein levels should be detected. Quantification of band intensities normalized to loading controls (e.g., β-actin) is essential .

• Immunofluorescence: Provides visual confirmation of ATP6V1G1 knockdown or overexpression at the cellular level, allowing assessment of expression levels in individual cells and subcellular localization patterns .

• Functional validation: Beyond confirming expression changes, researchers should validate functional consequences of ATP6V1G1 manipulation:

  • Measure V-ATPase activity using acidification assays

  • Assess the phosphorylation status of proteins known to be regulated by ATP6V1G1, such as RPS6, SQSTM1, PDPK1, and EEF2

  • Evaluate cellular phenotypes associated with ATP6V1G1 expression changes, such as proliferation, migration, and resistance to apoptosis

• Controls: Proper controls are essential:

  • For knockdown studies: use non-targeting siRNA/shRNA controls

  • For overexpression studies: use empty vector controls

  • Include positive controls such as GBM cell lines (LN229, T98G) known to express high levels of ATP6V1G1

Research has shown successful construction of HepG2 and Huh7 HCC cell lines with stable overexpression of ATP6V1G1 through lentiviral transfection, validated by both RT-PCR and western blot .

How can ATP6V1G1 antibodies be used to study its role in glioblastoma progression?

• Tissue microarray analysis: Perform immunohistochemistry using ATP6V1G1 antibodies on patient tumor samples of different grades to establish correlation between expression levels and tumor grade. Studies have shown that ATP6V1G1 expression is higher in high-grade human glioma tissues compared to grade II tumors .

• Cancer stem cell investigation: Analyze ATP6V1G1 expression in GBM neurospheres (cancer stem cell-enriched) compared to their differentiated counterparts using both western blotting and immunofluorescence. Research has demonstrated elevated gene and protein expression of ATP6V1G1 in neurospheres, which significantly decreases upon differentiation .

• Functional studies: After manipulating ATP6V1G1 expression in GBM cell lines, assess changes in:

  • Proliferation and colony formation

  • Migration and invasion

  • Apoptosis resistance

  • Response to standard GBM treatments

• Microenvironment interactions: Investigate how ATP6V1G1 expression influences the tumor microenvironment, as research suggests it may interact with other factors to reprogram the surrounding non-tumor microenvironment to a pro-tumor state .

• In vivo models: Validate findings using orthotopic GBM models with ATP6V1G1 knockdown or overexpression, followed by immunohistochemical analysis of tumor sections.

What methods can be used to investigate ATP6V1G1's involvement in hepatocellular carcinoma using specific antibodies?

Hepatocellular carcinoma (HCC) represents another cancer type where ATP6V1G1 plays a significant role in disease progression . Researchers can employ ATP6V1G1 antibodies in various methodological approaches to investigate this involvement:

• Expression analysis in patient samples: Perform immunohistochemistry on HCC tissue microarrays to correlate ATP6V1G1 expression with clinicopathological features and patient outcomes. Public data has revealed that ATP6V1G1 expression is dramatically increased in HCC compared to normal tissues and is elevated in advanced stages of HCC .

• Phosphoproteomic profiling: After manipulating ATP6V1G1 expression in HCC cell lines, use phospho-specific antibodies to validate changes in phosphorylation levels of key proteins:

  • Upregulated phosphoproteins: p-RPS6(Ser235) and p-SQSTM1(Ser272)

  • Downregulated phosphoproteins: p-PDPK1(Ser241) and p-EEF2(Ser57)

• Pathway analysis: Investigate the impact of ATP6V1G1 on critical signaling pathways in HCC:

  • mTOR and PI3K/Akt pathways, which involve RPS6 phosphorylation

  • Autophagy regulation through SQSTM1 phosphorylation

  • Protein synthesis regulation via EEF2 phosphorylation

• Functional validation: Assess the effects of ATP6V1G1 manipulation on:

  • HCC cell proliferation and colony formation

  • Migration and invasion capabilities

  • Resistance to apoptosis

  • Response to standard HCC treatments

• Mechanistic studies: Use co-immunoprecipitation with ATP6V1G1 antibodies to identify protein interaction partners that may mediate its effects on phosphorylation-related pathways in HCC.

How can ATP6V1G1 antibodies be used to investigate its role in neurological disorders?

ATP6V1G1 has been implicated in several neurological disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) . ATP6V1G1 antibodies can be valuable tools for investigating these connections:

• Expression analysis in patient samples: Perform immunohistochemistry and western blotting on post-mortem brain tissue from patients with ALS, FTD, and other neurodegenerative disorders to assess ATP6V1G1 expression levels compared to controls.

• Mutation studies: Investigate how UBQLN2 mutations, which have been found to cause ATP6V1G1 overexpression leading to ALS and FTD , affect ATP6V1G1 protein levels and localization using specific antibodies.

• Cell models: In neuronal cell cultures with manipulated ATP6V1G1 expression:

  • Assess lysosomal function and autophagy using co-immunofluorescence with lysosomal markers

  • Evaluate effects on proteostasis and protein aggregation

  • Investigate mitochondrial function and oxidative stress

• Animal models: In transgenic mouse models of neurodegenerative diseases:

  • Perform immunohistochemistry to analyze ATP6V1G1 expression patterns in different brain regions

  • Assess correlation between ATP6V1G1 levels and disease progression

  • Investigate rescue effects of normalizing ATP6V1G1 expression

• Development studies: Analyze ATP6V1G1 expression in embryonic neural tissues, as research has shown it is abundant in rat embryonic hippocampal neuronal cells (E18 RHN) , suggesting a role in neural development.

What are the optimal protocols for detecting phosphorylated proteins regulated by ATP6V1G1?

The detection of phosphorylated proteins regulated by ATP6V1G1 requires careful methodological considerations to ensure reliable and reproducible results. Based on available research, the following protocol has proven effective:

• Sample preparation:

  • Extract total proteins from cells using RIPA lysate containing cocktail protease phosphatase inhibitor and PMSF

  • Determine protein concentration using the BCA assay

  • For phosphoproteomic analysis, take 200μg protein for reduction and alkylation, followed by trypsin digestion overnight at 37°C

  • Label resulting peptides with TMT6 labeling reagent

• Phosphopeptide enrichment:

  • Enrich phosphorylated peptides using the TiO2 method

  • Separate by liquid chromatography

• Western blot validation:

  • Separate proteins by SDS-PAGE and transfer to PVDF membrane

  • Block membranes in TBST with 5% skim milk for 1 hour

  • Incubate with specific primary antibodies overnight at 4°C:

    • anti-p-RPS6 (Ser235) at 1:1000 dilution

    • anti-p-SQSTM1 (Ser272) at 1:1000 dilution

    • anti-p-EEF2 (Ser57) at 1:1000 dilution

    • anti-p-PDPK1 (Ser241) at 1:1000 dilution

    • anti-β-actin at 1:1000 dilution (loading control)

  • Incubate with secondary antibodies for 1 hour at room temperature

• Quantification and analysis:

  • Perform densitometric analysis of western blot bands

  • Normalize phosphoprotein levels to total protein levels when possible

  • Compare phosphorylation profiles between ATP6V1G1 overexpression/knockdown and control groups

This protocol has successfully identified 163 differentially expressed phosphorylated proteins with 228 altered phosphorylation sites in ATP6V1G1 overexpression studies .

How can researchers optimize immunofluorescence protocols for ATP6V1G1 detection in different cell types?

Optimizing immunofluorescence protocols for ATP6V1G1 detection requires consideration of cell type-specific factors and experimental goals. Based on published methodologies, the following optimization strategies are recommended:

• Fixation and permeabilization:

  • For most cell types, PFA fixation (4%) followed by Triton X-100 permeabilization has proven effective for ATP6V1G1 detection

  • For neurons or delicate primary cells, milder permeabilization with 0.1% Triton X-100 or 0.1% saponin may preserve cellular structures better

  • For membrane localization studies, avoid methanol fixation which can disrupt membrane structures

• Antibody concentration optimization:

  • Start with ATP6V1G1 antibodies at 4 μg/ml as successfully used for PC-3 cells

  • For primary cells or tissues, a concentration range of 1-10 μg/ml should be tested

  • General recommended dilution range for immunofluorescence is 1:50 to 1:200

• Cell type-specific considerations:

  • For cancer stem cells (neurospheres): optimize for suspension culture using low-speed centrifugation on poly-L-lysine coated slides

  • For highly confluent cultures: ensure adequate permeabilization time to allow antibody penetration

  • For co-staining with V-ATPase complex markers: carefully select compatible antibodies raised in different host species

• Signal amplification strategies:

  • For low expression contexts: consider tyramide signal amplification

  • For high background: increase blocking time or use alternative blocking reagents (5% BSA, normal serum)

  • For specific subcellular localization: combine with organelle markers (lysosomes, endosomes) for co-localization studies

• Validation controls:

  • Include positive controls (LN229, T98G glioblastoma cells)

  • Include negative controls (primary antibody omission, isotype controls)

  • For overexpression or knockdown studies, include appropriate genetic manipulation controls

What considerations are important when designing ATP6V1G1 knockdown experiments for functional studies?

Designing effective ATP6V1G1 knockdown experiments for functional studies requires careful planning to ensure specificity, efficiency, and proper phenotypic analysis. Based on research approaches, consider the following:

• Knockdown method selection:

  • siRNA transfection: suitable for short-term studies (48-72 hours)

  • shRNA via lentiviral transduction: preferable for stable, long-term knockdown studies

  • CRISPR-Cas9: for complete knockout studies where viable

• Target sequence design:

  • Design multiple target sequences to control for off-target effects

  • Ensure specificity for ATP6V1G1 by avoiding sequences with homology to ATP6V1G2 or ATP6V1G3

  • Target conserved regions if working with non-human models

• Validation of knockdown efficiency:

  • Validate at mRNA level using RT-PCR

  • Confirm protein reduction using western blotting with ATP6V1G1 antibody

  • Quantify knockdown efficiency through densitometric analysis

• Functional assays following knockdown:

  • V-ATPase activity: measure acidification using pH-sensitive dyes

  • Cell phenotype: assess proliferation, migration, invasion, and apoptosis

  • Pathway analysis: examine effects on phosphorylation of key proteins (RPS6, SQSTM1, PDPK1, EEF2)

  • Lysosomal function: evaluate autophagy flux and degradative capacity

• Control considerations:

  • Include non-targeting siRNA/shRNA controls

  • Consider rescue experiments by reintroducing ATP6V1G1 to confirm specificity

  • Include positive controls such as knockdown of known V-ATPase subunits

• Cell type-specific considerations:

  • For cancer stem cells: evaluate effects on stemness markers and differentiation capacity

  • For neurons: assess impact on synaptic function and neurodegeneration markers

  • For hepatocytes: examine effects on lipid metabolism

It's important to note that complete ATP6V1G1 knockdown may have profound effects on cell viability due to its crucial role in cellular pH homeostasis, so titrating knockdown levels or using inducible systems may be necessary for some experimental contexts.