PPP1R14A Antibody

What is PPP1R14A and what is its primary function in cellular physiology?

PPP1R14A, also known as CPI-17 (17 kDa PKC-potentiated inhibitory protein of PP1), functions as a phosphorylation-dependent inhibitor of smooth muscle myosin phosphatase. When phosphorylated, it exhibits over 1,000 times greater inhibitory activity, effectively serving as a molecular switch that regulates the phosphorylation state of PPP1CA substrate and smooth muscle contraction .

The inhibition of myosin phosphatase by phosphorylated PPP1R14A leads to increased myosin phosphorylation, enhancing smooth muscle contraction without requiring elevated intracellular Ca²⁺ concentrations. This mechanism is central to calcium sensitization, where PPP1R14A enables increases in myosin phosphorylation and tension at constant calcium levels .

How should PPP1R14A antibodies be stored to maintain optimal activity?

For maximum stability and retention of antibody activity, PPP1R14A antibodies should be stored at -20°C or lower temperatures . To prevent protein degradation from repeated freeze-thaw cycles, it is recommended to aliquot the antibody solution before freezing . When properly stored, most commercial PPP1R14A antibodies maintain their efficacy for approximately 12 months from the date of shipment .

What types of PPP1R14A antibodies are available for research, and how do they differ?

Current research-grade PPP1R14A antibodies are available in multiple formats with distinct characteristics:

Mouse monoclonal antibodies offer high specificity and reproducibility between experimental batches. The 3A7 clone has been validated for western blotting and ELISA applications, with a detection limit for recombinant GST-tagged PPP1R14A of approximately 0.1ng/ml when used as a capture antibody . Rabbit monoclonal antibodies like E305 have been successfully used in western blot applications at dilutions of 1:2500 in mouse samples .

How does the expression pattern of PPP1R14A differ between normal and cancerous tissues, and what are the implications for antibody-based detection methods?

PPP1R14A exhibits a complex expression pattern across cancer types that challenges conventional tumor marker paradigms. Comprehensive pan-cancer analysis reveals that PPP1R14A is predominantly downregulated in major malignancies compared to matched normal tissues . This downregulation pattern is particularly notable in bladder urothelial carcinoma (BLCA), colon adenocarcinoma (COAD), and kidney renal papillary cell carcinoma (KIRP) .

For antibody-based detection methods, these findings necessitate:

• Careful selection of control tissues that match the cancer stage being studied

• Interpretation of immunohistochemistry results with consideration of this dual role

• Potential use of PPP1R14A antibodies for diagnostic purposes, as ROC analysis indicates excellent diagnostic accuracy (AUC > 0.9) for multiple malignancies including BLCA, COAD, and KIRP

What methodological approaches can resolve apparently contradictory results when studying PPP1R14A expression in different cancer contexts?

Resolving contradictory PPP1R14A expression patterns requires a multi-dimensional analytical framework:

• Temporal dimension analysis: Separate studies of cancer initiation versus progression stages to delineate the transitional patterns of PPP1R14A expression. Evidence shows that while PPP1R14A is generally downregulated in tumor versus normal tissue comparison, its expression increases as cancer progresses in many cancer types .

• Integrated multi-omics approach: Combine transcriptomic data with:

  • Methylation analysis: PPP1R14A promoter hypermethylation occurs in most common cancers, consistent with its initial downregulation

  • Phosphorylation studies: Examine phosphorylation levels at sites including S26 and T38, which show downregulation in breast cancer and colon cancer

  • Genetic alteration analysis: Assess copy number alterations and mutations, which reach 16.07% frequency in uterine carcinosarcoma and affect patient prognosis

• Context-specific validation: For instance, in cholangiocarcinoma (CHOL) and head and neck squamous cell carcinoma (HNSC), high promoter methylation correlates with high transcription levels rather than suppression, suggesting non-canonical regulatory mechanisms that merit targeted investigation .

When designing antibody-based detection experiments, researchers should incorporate appropriate controls for each cancer type and stage, and validate findings using multiple methodological approaches.

What technical considerations are critical when using PPP1R14A antibodies for western blot analysis?

Successful western blot analysis using PPP1R14A antibodies requires attention to several technical parameters:

• Molecular weight interpretation: PPP1R14A detection can show varying molecular weights dependent on experimental conditions:

  • The native protein is expected at approximately 16.7 kDa in transfected lysates

  • The immunogen used for antibody production may be detected at 41.91 kDa

  • Post-translational modifications, particularly phosphorylation states, can alter migration patterns

• Antibody selection and dilution optimization:

  • Mouse monoclonal antibodies such as F-4 (Santa Cruz Biotechnologies, sc-48406) have been validated for mouse samples

  • Rabbit monoclonal antibody E305 (Abcam, ab32213) has been validated at 1:2500 dilution for mouse samples

  • Detection sensitivity varies by clone; the 3A7 clone can detect GST-tagged PPP1R14A at concentrations as low as 0.1ng/ml

• Control selection: Include both positive and negative controls:

  • PPP1R14A-transfected cell lysates serve as positive controls

  • Non-transfected lysates provide appropriate negative controls

  • For cancer studies, matched normal and tumor tissues at defined stages should be included to account for the complex expression dynamics

How can phosphorylation states of PPP1R14A be effectively studied, and what antibodies are suitable for this purpose?

Studying PPP1R14A phosphorylation states is crucial given that phosphorylation increases its inhibitory activity over 1,000-fold and functions as a molecular switch in signaling pathways . Methodological approaches include:

• Phospho-specific antibodies: While not explicitly mentioned in the search results, phospho-specific antibodies targeting key sites such as S26 and T38 would be essential for direct detection of phosphorylation states. These sites show altered phosphorylation levels in multiple cancer types .

• Phosphatase treatment controls: Samples can be split and treated with/without phosphatases prior to western blotting with total PPP1R14A antibodies to determine the proportion of phosphorylated protein.

• Phos-tag™ SDS-PAGE: This technique allows separation of phosphorylated and non-phosphorylated forms of proteins in acrylamide gels containing Phos-tag™ reagent, followed by detection with total PPP1R14A antibodies.

• Mass spectrometry: For comprehensive phosphorylation site mapping, immunoprecipitation with PPP1R14A antibodies followed by mass spectrometry analysis provides detailed insight into multiple phosphorylation sites simultaneously.

• Kinase/phosphatase assays: In vitro assays using purified PPP1R14A and relevant kinases (particularly PKC) or phosphatases, detected with total PPP1R14A antibodies, can establish phosphorylation/dephosphorylation kinetics.

How can PPP1R14A antibodies be utilized to study its contradictory roles in cancer initiation versus progression?

The dual role of PPP1R14A in cancer biology necessitates sophisticated experimental designs:

What is the relationship between PPP1R14A methylation status and protein expression, and how can this be investigated?

The relationship between PPP1R14A promoter methylation and expression presents a complex regulatory landscape:

The data from these integrated analyses can help establish predictive models for when methylation will lead to silencing versus potential activation of PPP1R14A, informing both diagnostic applications of PPP1R14A antibodies and therapeutic strategies targeting epigenetic mechanisms.

How can PPP1R14A antibodies be used to investigate its role in immune infiltration and potential immunotherapy applications?

PPP1R14A has been found to correlate significantly with levels of immune infiltrating cells and immune checkpoint genes , suggesting potential roles in tumor immunology. Methodological approaches include:

• Multiplex immunofluorescence:

  • Co-stain tissue sections with PPP1R14A antibodies and markers for specific immune cell populations (CD8+ T cells, macrophages, etc.)

  • Quantify spatial relationships between PPP1R14A-expressing tumor cells and immune infiltrates

  • Correlate patterns with response to immunotherapy in retrospective patient cohorts

• Flow cytometry:

  • Dissociate tumors into single-cell suspensions

  • Use PPP1R14A antibodies in combination with immune cell markers to quantify relationships between PPP1R14A expression and immune cell proportions

  • Sort cells based on PPP1R14A expression for downstream functional assays

• Co-immunoprecipitation:

  • Use PPP1R14A antibodies to pull down protein complexes

  • Analyze interacting partners using mass spectrometry to identify potential interactions with immune signaling components

  • Confirm interactions using reciprocal co-IP and western blotting

• In vitro co-culture systems:

  • Establish tumor cell lines with modulated PPP1R14A expression using CRISPR/Cas9 or siRNA

  • Co-culture with immune cells (T cells, macrophages)

  • Assess immune cell activation, cytokine production, and tumor cell killing

  • Use PPP1R14A antibodies to monitor expression in fixed and permeabilized cells via flow cytometry

• Correlation with immunotherapy response biomarkers:

  • Analyze relationship between PPP1R14A expression (detected by antibodies) and established biomarkers like PD-L1, tumor mutational burden, and T cell infiltration

  • Stratify patient cohorts by PPP1R14A expression to retrospectively evaluate immunotherapy response rates

What are common pitfalls when using PPP1R14A antibodies in experimental systems, and how can they be addressed?

Researchers should be aware of several technical challenges when working with PPP1R14A antibodies:

• Size discrepancies in western blots:

  • Expected size of native PPP1R14A is approximately 16.7 kDa

  • Some experiments detect bands at approximately 41.91 kDa (immunogen size)

  • Solution: Always include positive controls (PPP1R14A-transfected lysates) and negative controls (non-transfected lysates) to establish the correct band pattern for your experimental system

• Phosphorylation state influence:

  • PPP1R14A function is heavily dependent on phosphorylation status

  • Solution: Consider using phospho-specific antibodies or Phos-tag™ gels to distinguish phosphorylated forms

• Cancer context complexity:

  • Expression patterns differ dramatically between cancer initiation and progression

  • Solution: Clearly define the cancer stage being studied and interpret results within the appropriate context

• Cross-reactivity concerns:

  • PPP1R14A belongs to a family of related inhibitor proteins

  • Solution: Validate antibody specificity using knockout/knockdown controls or peptide competition assays

• Fixation sensitivity in immunohistochemistry:

  • Different fixation methods may affect epitope accessibility

  • Solution: Optimize antigen retrieval methods and validate with multiple antibody clones when possible

How should researchers select the most appropriate PPP1R14A antibody clone for their specific application?

Selection of the optimal PPP1R14A antibody clone should be guided by the following considerations:

• Application compatibility:

  • For western blotting: Both mouse monoclonal (F-4, 3A7) and rabbit monoclonal (E305) antibodies have been validated

  • For ELISA: The 3A7 clone has demonstrated efficacy as a capture antibody with sensitivity to 0.1ng/ml

• Species cross-reactivity:

  • For human samples: Mouse monoclonal antibodies targeting human PPP1R14A (AAH21089) are available

  • For mouse samples: Both rabbit monoclonal (E305) and mouse monoclonal (F-4) antibodies have been validated

• Epitope accessibility:

  • Consider the structural context of your experiment (native vs. denatured protein)

  • Review the immunogen sequence used to generate the antibody to ensure epitope relevance

• Published validation:

  • The rabbit monoclonal E305 (Abcam, ab32213) has been validated in mouse samples at 1:2500 dilution

  • Mouse monoclonal F-4 (Santa Cruz Biotechnologies, sc-48406) has been validated in mouse samples

• Clone-specific characteristics:

  • 3A7 clone: Mouse monoclonal IgG1 kappa targeting human PPP1R14A

  • E305 clone: Rabbit monoclonal validated for western blot applications

  • F-4 clone: Mouse monoclonal validated for western blot applications

Where possible, preliminary testing of multiple antibody clones is recommended to identify the optimal reagent for your specific experimental system.

How might PPP1R14A antibodies contribute to developing targeted cancer therapies?

PPP1R14A antibodies could facilitate the development of targeted cancer therapies through several research pathways:

• Biomarker development for precision medicine:

  • PPP1R14A antibodies can help stratify patients based on expression levels

  • ROC analysis indicates excellent diagnostic accuracy (AUC > 0.9) for several malignancies

  • Expression patterns could potentially predict treatment response or resistance mechanisms

• Target validation for drug development:

  • Antibody-based screening can identify cancer types with aberrant PPP1R14A function

  • Immunoprecipitation followed by mass spectrometry can reveal cancer-specific interacting partners

  • These interactions may represent novel therapeutic targets

• Monitoring therapy response:

  • Serial biopsies analyzed with PPP1R14A antibodies can track expression changes during treatment

  • Changes in phosphorylation status may serve as pharmacodynamic markers

• Potential antibody-drug conjugate (ADC) development:

  • For cancers where PPP1R14A is significantly upregulated during progression, modified therapeutic antibodies targeting surface-exposed epitopes could deliver cytotoxic payloads

  • Cancer type-specific expression patterns would need to be carefully validated

• Combination therapy rational design:

  • Given PPP1R14A's association with immune infiltration , antibody-based studies can help identify optimal immunotherapy combinations

  • Integration with phosphorylation data can inform combinations with kinase inhibitors

What novel methodological approaches might enhance PPP1R14A antibody applications in future research?

Emerging technologies offer opportunities to expand PPP1R14A antibody applications:

• Single-cell analysis:

  • Single-cell western blotting or CyTOF with PPP1R14A antibodies can reveal heterogeneity within tumors

  • This approach may help resolve seemingly contradictory bulk tissue findings regarding expression patterns

• Spatial transcriptomics integration:

  • Combining immunohistochemistry using PPP1R14A antibodies with spatial transcriptomics can map expression patterns within the tumor microenvironment

  • This integrative approach could reveal functional niches where PPP1R14A plays critical roles

• Organoid-based functional studies:

  • Patient-derived organoids representing different cancer stages can be analyzed with PPP1R14A antibodies

  • This system allows for controlled manipulation of PPP1R14A expression or phosphorylation to assess functional consequences

• Intravital imaging:

  • Development of fluorescently labeled PPP1R14A antibody fragments for in vivo imaging

  • This approach could enable real-time tracking of expression changes during tumor evolution

• Proximity labeling proteomics:

  • Fusion of PPP1R14A with proximity labeling enzymes (BioID, APEX) followed by detection with antibodies

  • This technique can map the PPP1R14A interactome in living cells under physiologically relevant conditions