PPP6R3 Antibody

What is PPP6R3 and what role does it play in cellular signaling?

PPP6R3, also known as SAPS3 (SAPS domain family member 3), functions as a regulatory subunit of protein phosphatase 6 (PP6). It serves as a scaffolding subunit that guides PP6 enzymatic activity to specific cellular events and plays a crucial role in determining PP6 substrate specificity . PPP6R3 is part of the PP6 holoenzyme complex that includes the catalytic subunit PP6C and other regulatory components. This complex is involved in various cellular processes including DNA repair mechanisms through dephosphorylating proteins such as H2AX, which are essential in sensing and repairing DNA damage .

What are the alternative nomenclature and identifiers for PPP6R3?

PPP6R3 is known by several alternative names in scientific literature:

• SAPS3 (SAPS domain family member 3)

• PP6R3 (Protein Phosphatase 6 Regulatory Subunit 3)

• SAPL (Sporulation-induced transcript 4-associated protein SAPL)

• C11orf23 (Chromosome 11 open reading frame 23)

• KIAA1558

• SAP190

In mouse models, the gene is designated as Ppp6r3 with Entrez ID: 52036 and UniProt ID: Q922D4 .

What is the tissue-specific expression pattern of PPP6R3?

PPP6R3 shows a complex tissue distribution pattern:

How does PPP6R3 regulate AMPK signaling pathways?

Recent research has revealed that PPP6R3/SAPS3 functions as a negative regulator of AMP-activated protein kinase (AMPK). The mechanism involves:

• Direct binding of SAPS3 to AMPK, demonstrated through recombinant protein studies

• Facilitation of PP6C (catalytic subunit) binding to AMPK, enabling dephosphorylation of AMPK and its downstream substrate ACC

• SAPS3 upregulation upon overnutrition, promoting AMPK inactivation

Knockout studies have shown that loss of SAPS3 leads to AMPK activation in vivo and maintains glucose homeostasis under high-fat diet conditions. This suggests that targeting SAPS3 might be a strategy to restore metabolic homeostasis in metabolic disorders .

What distinguishes PPP6R3 from other SAPS family members in functional studies?

Among the three SAPS family members (SAPS1, SAPS2, and SAPS3), only knockdown of SAPS3 was able to modulate AMPK dephosphorylation upon glucose readdition in experimental models . This functional specificity is important when designing experiments targeting particular PP6 regulatory pathways. While all three SAPS proteins are PP6 regulatory subunits, they exhibit different substrate specificities and cellular functions, making PPP6R3/SAPS3 uniquely associated with certain signaling pathways, particularly those involving AMPK regulation and metabolic homeostasis .

What is the relationship between PPP6R3 and immune function?

PPP6R3 appears to have important roles in immune cell function:

• It may play an important role in maintaining immune self-tolerance

• The highest expression levels of PP6c and SAPS subunits (including PPP6R3) are found in cells of the hematopoietic system

• Conditional knockout studies of PP6c in mice (Pp6c f/f lck-cre) showed impaired development of α/β-lineage thymocytes with decreased numbers in CD4+CD8+ double-positive and CD4+ and CD8+ single-positive compartments

• PPP6R3 was also identified in proteomics analysis as part of the TNF-α/NF-κB pathway, suggesting a role in inflammatory signaling

What validation criteria should be applied when selecting a PPP6R3 antibody?

When selecting a PPP6R3 antibody, researchers should consider multiple validation criteria:

• Specificity validation: Verify antibody specificity using tissues known to express PPP6R3 positively and negatively . Knockout or knockdown controls are essential to ensure the antibody does not detect off-target proteins of similar molecular weight .

• Application-specific validation: Confirm the antibody has been validated for your specific application (WB, IHC, IF, etc.) .

• Species reactivity: Check that the antibody has been validated in your species of interest. Common PPP6R3 antibodies show reactivity to human, mouse, rat, and other mammalian species .

• Epitope information: Consider the epitope location. Some antibodies target specific regions like the C-terminal domain, which may affect detection depending on protein interactions or post-translational modifications .

• Published validation: Look for antibodies cited in peer-reviewed publications with appropriate validation data .

What applications are PPP6R3 antibodies commonly validated for?

PPP6R3 antibodies have been validated for several research applications:

• Western Blotting (WB): Most commercially available PPP6R3 antibodies are validated for western blot applications, detecting bands around 98-120 kDa .

• Immunohistochemistry (IHC): Some antibodies are validated for IHC on paraffin-embedded (IHC-P) and frozen (IHC-fro) tissue sections .

• Immunofluorescence (IF): Several antibodies can be used for immunocytochemistry (ICC) and immunofluorescence on cultured cells (IF-cc) or tissue sections (IF-p) .

• Immunoprecipitation (IP): Certain antibodies are validated for immunoprecipitation studies to investigate protein-protein interactions .

• ELISA: Some antibodies are validated for enzyme-linked immunosorbent assay applications .

How should researchers interpret discrepancies in PPP6R3 detection between different antibodies?

When facing discrepancies in PPP6R3 detection between different antibodies, researchers should consider:

• Epitope accessibility: Different antibodies may target different epitopes that could be masked by protein-protein interactions or post-translational modifications in certain cellular contexts.

• Antibody specificity issues: As highlighted in research on phospho-specific antibodies, seemingly specific antibodies may detect unmodified forms of proteins or be sensitive to modifications at neighboring residues .

• Validation approach: The validation method used by manufacturers may not match your experimental conditions. For example, an antibody validated in western blotting may not perform similarly in immunohistochemistry.

• Isoform detection: Ensure the antibody recognizes the specific isoform of interest in your experimental system.

To address these discrepancies:

• Use multiple antibodies targeting different epitopes

• Perform knockdown/knockout experiments as controls

• Include positive and negative control tissues based on known expression patterns

• Consider the molecular weight of detected bands (expected ~98-120 kDa)

What are the optimal western blotting conditions for detecting PPP6R3?

For optimal western blotting detection of PPP6R3:

• Sample preparation: Prepare whole cell lysates using standard protocols. As demonstrated in validation studies, whole cell lysates from HeLa or 293T cells serve as good positive controls .

• Loading amount: Load appropriate protein amounts (typically 5-50 μg of whole cell lysate) .

• Expected molecular weight: Look for bands at approximately 98-120 kDa. The predicted molecular weight is 98 kDa, but observed bands may appear around 120 kDa due to post-translational modifications .

• Antibody dilution: Follow manufacturer recommendations, but typical working dilutions range from 0.02-0.04 μg/mL for highly sensitive antibodies .

• Detection system: Use standard chemiluminescence detection systems appropriate for rabbit polyclonal antibodies, as most PPP6R3 antibodies are rabbit-derived .

• Validation controls: Include positive controls (tissues/cells with known PPP6R3 expression) and consider siRNA knockdown samples as negative controls.

How can researchers effectively use PPP6R3 antibodies to study protein-protein interactions?

To effectively study PPP6R3 protein-protein interactions:

• Co-immunoprecipitation: PPP6R3 antibodies validated for IP can be used to pull down PPP6R3 and associated proteins. For example, studies have used co-IP/co-transfection to validate the interaction between SAPS3 and AMPK in 293T cells .

• Reciprocal co-IP: Perform reciprocal pull-downs with antibodies against potential interacting partners (e.g., PP6C, AMPK) to confirm specific interactions.

• Direct binding assays: Use recombinant proteins to assess direct binding in vitro, as demonstrated in studies showing direct binding between SAPS3 and AMPK .

• Subcellular co-localization: Use immunofluorescence with PPP6R3 antibodies to examine co-localization with potential interacting proteins. Studies have shown endogenous SAPS3 co-localizes with AMPK in certain cell types .

• Functional validation: Complement interaction studies with functional assays that measure the impact of PPP6R3 knockdown on the activity of interacting proteins (e.g., AMPK phosphorylation status) .

What considerations are important when using PPP6R3 antibodies in immunohistochemistry?

When using PPP6R3 antibodies for immunohistochemistry:

• Tissue selection: Consider the known expression pattern of PPP6R3, with highest protein levels in lung, spleen, bladder, and liver for positive controls .

• Antibody validation: Choose antibodies specifically validated for IHC-P or IHC-frozen applications .

• Antigen retrieval: Optimize antigen retrieval methods, as epitope accessibility may be affected by fixation and embedding processes.

• Signal specificity: Validate signal specificity using tissues from knockdown/knockout models or comparing with known expression patterns.

• Detection system: Select appropriate secondary antibodies and detection systems compatible with the host species of the primary antibody (typically rabbit for PPP6R3 antibodies) .

• Quantification approaches: For expression analyses, develop consistent scoring methods based on staining intensity and distribution patterns.

How can researchers address antibody cross-reactivity issues when studying PPP6R3?

To address potential cross-reactivity with PPP6R3 antibodies:

• Genetic validation: Use CRISPR/Cas9 knockout or siRNA knockdown models to confirm antibody specificity. This is especially important as studies have shown that even well-characterized antibodies can have cross-reactivity issues .

• Peptide competition: Perform peptide competition assays where the antibody is pre-incubated with the immunizing peptide before application to verify specific binding.

• Multiple antibodies approach: Use multiple antibodies targeting different epitopes of PPP6R3 and compare results.

• Western blot validation: Confirm a single band of expected molecular weight (98-120 kDa) before using the antibody in other applications .

• Tissue panel validation: Test the antibody on multiple tissues with known expression patterns of PPP6R3 to confirm detection correlates with expected expression levels .

What are common technical pitfalls when working with phosphatase regulatory subunit antibodies?

Common pitfalls with phosphatase regulatory subunit antibodies include:

• Post-translational modification interference: Antibody detection may be affected by phosphorylation, methylation, or other modifications of the target protein or neighboring residues, as shown in studies with phospho-specific antibodies .

• Isoform specificity: Ensure the antibody detects all relevant isoforms or specifically the isoform of interest.

• Protein complex masking: Epitopes may be masked when the regulatory subunit is in complex with catalytic subunits or other proteins.

• Antibody validation assumptions: Don't assume an antibody validated for one application will work for others without specific validation .

• Signal-to-noise ratio: Optimize blocking conditions, antibody dilutions, and washing steps to improve signal-to-noise ratio, especially for less abundant regulatory subunits.

How should inconsistencies between mRNA and protein expression data for PPP6R3 be interpreted?

Research has demonstrated significant discordance between mRNA and protein expression patterns for PPP6R3 across different tissues . When interpreting such inconsistencies:

• Post-transcriptional regulation: Consider that PPP6R3 expression is likely regulated post-transcriptionally, at the level of mRNA translation or protein stability .

• Tissue-specific mechanisms: Different tissues may employ distinct regulatory mechanisms. For example, PPP6R3 mRNA is highest in heart, but protein levels are highest in lung, bladder, spleen, and pancreas .

• Experimental approach validation: Use multiple techniques to measure both mRNA (qRT-PCR, Northern blot) and protein (Western blot, IHC, mass spectrometry) levels to confirm findings.

• Functional relevance: Focus on protein levels for functional studies, as these ultimately determine biological activity, while considering mRNA levels for understanding transcriptional regulation.

• Temporal dynamics: Consider that mRNA and protein may have different temporal dynamics in response to stimuli or during development.

How can PPP6R3 antibodies be utilized to study metabolic regulation pathways?

PPP6R3/SAPS3 has recently been identified as an AMPK inhibitor with significant implications for metabolic regulation . Researchers can utilize PPP6R3 antibodies to:

• Monitor expression changes: Assess how PPP6R3 levels change in response to metabolic stimuli or disease states. Research has shown SAPS3 is upregulated upon overnutrition .

• Track protein interactions: Use co-immunoprecipitation with PPP6R3 antibodies to examine dynamic interactions with AMPK and PP6C under different metabolic conditions .

• Assess phosphorylation status: Combine PPP6R3 antibodies with phospho-specific antibodies against AMPK to correlate PPP6R3 levels with AMPK activity.

• Tissue-specific expression: Map PPP6R3 expression across metabolically active tissues in normal and disease states using immunohistochemistry.

• Therapeutic target validation: Evaluate PPP6R3 as a potential therapeutic target in metabolic disorders by correlating expression with disease markers and metabolic parameters in clinical samples.

What role might PPP6R3 play in cancer research and how can antibodies facilitate this investigation?

While the search results don't directly address PPP6R3 in cancer, several aspects suggest potential relevance:

• Cell cycle regulation: As a phosphatase regulatory subunit, PPP6R3 may influence cell cycle progression through phosphorylation/dephosphorylation events.

• DNA damage response: PPP6R3/SAPS3 interacts with DNA repair mechanisms through dephosphorylation of proteins like H2AX , which may have implications for genomic stability in cancer.

• Metabolic reprogramming: Given its role in AMPK regulation , PPP6R3 may influence metabolic reprogramming in cancer cells.

• NF-κB signaling: PPP6R3's involvement in the TNF-α/NF-κB pathway suggests potential roles in inflammation-associated cancers.

Researchers can use PPP6R3 antibodies to:

• Analyze expression patterns across cancer types and stages

• Correlate expression with patient outcomes

• Investigate subcellular localization changes during malignant transformation

• Study protein interactions specific to cancer contexts

What emerging technologies might enhance the specificity and applications of PPP6R3 antibodies?

Emerging technologies that could enhance PPP6R3 antibody applications include:

• Nanobodies and single-domain antibodies: These smaller antibody fragments may provide better access to epitopes in complex proteins or protein complexes.

• Proximity labeling techniques: BioID or APEX2 fusions with PPP6R3 could provide insights into the dynamic interactome of PPP6R3 in different cellular contexts.

• Super-resolution microscopy: Combined with highly specific antibodies, techniques like STORM or PALM could reveal the precise subcellular localization and co-localization patterns of PPP6R3.

• Recombinant antibody engineering: Creating recombinant antibodies with enhanced specificity and reduced cross-reactivity through in vitro evolution and selection methods.

• Mass cytometry (CyTOF): Integrating PPP6R3 antibodies into CyTOF panels could allow simultaneous analysis of multiple signaling pathways in single cells.

• CRISPR knock-in tags: Endogenous tagging of PPP6R3 could provide alternatives to antibody-based detection while maintaining physiological expression levels.