PPP2CA Antibody

What is PPP2CA and what cellular functions does it regulate?

PPP2CA is the alpha isoform of the catalytic subunit of protein phosphatase 2A (PP2A), a serine/threonine phosphatase that regulates multiple cellular pathways. Recent studies indicate that PPP2CA plays critical roles in epithelial-to-mesenchymal transition (EMT), cancer progression, and immune cell function . The protein has a molecular weight of approximately 36 kDa and consists of 309 amino acids . PPP2CA activity modulates key signaling pathways including Akt, β-catenin, and NF-κB, impacting cell survival, proliferation, and differentiation . Additionally, recent phosphoproteomic studies have identified over 2,200 phosphoproteins as potential PPP2CA substrates, with roles in spliceosomes, RNA transport, and cell cycle regulation .

How should I select the appropriate PPP2CA antibody for my experiments?

Antibody selection should be guided by your specific application requirements:

For specialized applications like cytometric bead arrays, consider recombinant antibodies (e.g., 84155-2-PBS) that offer consistent batch-to-batch performance . Always verify reactivity with your species of interest, as some antibodies are human-specific while others cross-react with mouse and rat samples .

What control samples should I include when validating a PPP2CA antibody?

Proper controls are essential for antibody validation:

• Positive controls: Use cells with documented PPP2CA expression such as HEK-293T, Jurkat, or MCF-7 cells . Human liver tissue has also been validated for IHC applications .

• Negative controls:

  • PPP2CA knockdown or knockout samples using siRNA, shRNA, or CRISPR/Cas9 technology

  • Secondary antibody-only controls to assess non-specific binding

  • Isotype controls for techniques like IP and flow cytometry

• Specificity controls:

  • Blocking peptide competition assays

  • Detection of expected molecular weight (36 kDa)

  • Multiple antibodies targeting different epitopes to confirm patterns

Recent studies have successfully generated PPP2CA-deficient models, including CRISPR/Cas9 knockouts in neuroblastoma cells and conditional knockouts in T cells, which provide excellent negative controls .

What are the optimal conditions for detecting PPP2CA by Western blotting?

For successful Western blot detection of PPP2CA:

• Sample preparation:

  • Include phosphatase inhibitors in lysis buffers to preserve phosphorylation states

  • Standardize protein loading (20-50 μg total protein per lane)

  • Denature samples at 95°C for 5 minutes in reducing conditions

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal resolution around 36 kDa

  • Semi-dry or wet transfer systems are both suitable

  • Verify transfer efficiency with Ponceau S staining

• Antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST

  • Dilute primary antibody 1:1000-1:4000 in blocking buffer

  • Incubate overnight at 4°C for optimal signal-to-noise ratio

  • Use HRP-conjugated secondary antibodies (typically 1:5000-1:10000)

• Detection systems:

  • Enhanced chemiluminescence (ECL) reagents work well for PPP2CA detection

  • WESTAR ETA ULTRA reagent has been used successfully in published studies

  • For quantitative analysis, consider digital imaging systems

Published studies have successfully detected PPP2CA in various sample types including HEK-293T cells, Jurkat cells, MCF-7 cells, and human liver tissue .

How can I optimize immunofluorescence staining for PPP2CA?

For high-quality immunofluorescence imaging of PPP2CA:

• Cell preparation:

  • Culture cells on glass coverslips or chamber slides

  • Fix with 4% paraformaldehyde (10-15 minutes at room temperature)

  • Permeabilize with 0.1-0.3% Triton X-100

• Antibody staining:

  • Block with 5-10% normal serum from secondary antibody species

  • Dilute primary antibody 1:50-1:500 in blocking buffer

  • Incubate overnight at 4°C or 1-2 hours at room temperature

  • Use fluorophore-conjugated secondary antibodies at 1:200-1:1000

• Mounting and imaging:

  • Mount with anti-fade medium containing DAPI for nuclear counterstaining

  • Use confocal microscopy for precise subcellular localization

  • Adjust exposure to avoid saturation while maintaining signal visibility

• Controls and validation:

  • Include secondary-only controls

  • Use cells with validated PPP2CA expression (e.g., MCF-7 cells)

  • Consider co-staining with markers of relevant subcellular compartments

MCF-7 cells have been validated for PPP2CA immunofluorescence and can serve as a reliable positive control .

What strategies can I use to study PPP2CA substrates?

Modern approaches to identify and validate PPP2CA substrates include:

• Targeted degradation coupled with phosphoproteomics:

  • Generate dTAG/dTAG PPP2CA homozygous knock-in cells using CRISPR/Cas9

  • Use dTAG-13 PROTAC for selective and efficient degradation of endogenous PPP2CA

  • Perform quantitative phosphoproteomics to identify affected phosphosites

This approach has revealed 2,204 phosphoproteins as putative PPP2CA substrates, with prominent pSP/pTP motifs as preferred dephosphorylation targets .

• Genetic manipulation approaches:

  • Compare phosphoproteomes in PPP2CA-deficient versus wild-type cells

  • Recent studies identified significant changes in phosphorylation of proteins like RPS6, AKT, and STAT5 in T cells lacking PPP2CA

  • Ingenuity pathway analysis of phosphoproteomic data identified PI3K/AKT and EIF2 signaling as top pathways affected by PPP2CA deficiency

• Biochemical validation:

  • In vitro dephosphorylation assays with recombinant PPP2CA

  • Site-directed mutagenesis of candidate phosphosites

  • Phospho-specific antibodies to monitor dephosphorylation kinetics

These complementary approaches provide robust identification and validation of physiologically relevant PPP2CA substrates.

How does PPP2CA expression affect cancer progression and what experimental models can address this?

PPP2CA has emerged as a critical regulator of cancer behavior:

• Expression patterns and clinical correlations:

  • In hepatocellular carcinoma, PPP2CA expression correlates with Child-Pugh classification, AST levels, microvascular invasion, and portal cancer thrombus

  • PPP2CA downregulation is associated with aggressive prostate cancer phenotypes and castration resistance

• Experimental models to study PPP2CA in cancer:

  • Cell line models:

    • Overexpression in low-expressing lines (C4-2, PC3)

    • Silencing in high-expressing lines (LNCaP)

    • CRISPR/Cas9 knockout (e.g., in KELLY neuroblastoma cells)

  • In vivo models:

    • Orthotopic xenografts with PPP2CA-manipulated cells

    • Conditional knockout mouse models

    • Patient-derived xenografts with varying PPP2CA expression

• Functional assays:

  • Migration and invasion assays have demonstrated that PPP2CA-overexpressing C4-2 and PC3 cells show significantly reduced migratory (2.3- and 2.2-fold) and invasive (2.7- and 2.8-fold) potential

  • Conversely, PPP2CA-knockdown LNCaP cells exhibit enhanced migration (2.4-fold) and invasion (3.0-fold)

  • Animal studies have confirmed a suppressive effect of PPP2CA expression on prostate cancer growth and metastasis

• Mechanistic insights:

  • PPP2CA restoration decreases nuclear accumulation and transcriptional activity of β-catenin and NF-κB

  • PPP2CA regulates Akt and ERK phosphorylation, which are elevated in PPP2CA-silenced cells

  • PPP2CA loss promotes epithelial-to-mesenchymal transition, contributing to cancer invasiveness

These findings suggest PPP2CA as both a prognostic biomarker and potential therapeutic target in multiple cancer types.

What is the role of PPP2CA in immune cell function and how can it be experimentally assessed?

Recent research has uncovered critical functions of PPP2CA in immune responses:

• T-cell development and function:

  • PPP2CA is essential for CD8+ T-cell responses to infection

  • Cd4-Cre Ppp2ca-deficient mice show impaired antigen-specific CD8+ T-cell expansion and reduced production of IFN-γ and TNF-α following Listeria monocytogenes-OVA infection

  • These defects persist even with Bcl2 transgene expression, indicating they are independent of survival defects

• Experimental approaches:

  • Genetic models: Conditional knockout using Cd4-Cre Ppp2ca^f/f mice

  • Chimera models: Bone marrow transplantation to distinguish cell-intrinsic effects

  • Ex vivo stimulation: Anti-CD3/anti-CD28 activation of isolated T cells

  • Flow cytometry: Analysis of T-cell activation markers and cytokine production

  • Infection models: Listeria monocytogenes-OVA challenge to assess antigen-specific responses

• Molecular mechanisms:

  • Proteomic analysis revealed that PPP2CA deficiency destabilizes the entire PP2A holoenzyme complex, with reduced expression of multiple PP2A components (PPP2CB, PPP2R5A, PPP2R5B, PPP2R1A, PPP2R5C)

  • Phosphoproteomic studies identified increased S6 phosphorylation (indicating mTORC1 activation) and altered AKT and STAT5 phosphorylation in PPP2CA-deficient T cells

  • Pathway analysis highlighted disruptions in PI3K/AKT signaling and EIF2 pathways

These findings suggest that PPP2CA status may influence immunotherapy outcomes and could potentially be targeted to enhance T-cell responses in cancer and infectious disease.

What combination strategies involving PPP2CA show promise in therapeutic research?

Emerging research highlights potential therapeutic approaches involving PPP2CA:

• PP2A activators:

  • Several small molecules that activate PP2A have been developed

  • These could potentially restore PPP2CA function in cancers where it is downregulated

• Combination therapies:

  • Researchers have explored combining PP2A modulation with kinase inhibitors

  • The combination index (CI) can be calculated using the Chou-Talalay method to determine synergistic effects

  • Weighted average CI values (CIwt) are calculated as:
    CIwt = (CI50 + 2CI75 + 3CI90 + 4CI95)/10

  • This approach helps identify optimal drug combinations and dosing strategies

• CRISPR/Cas9-based approaches:

  • PPP2CA gene ablation using CRISPR/Cas9 has been employed to assess therapeutic potential

  • For example, KELLY neuroblastoma cell line was selected for such studies as a paradigmatic model

• Experimental design considerations:

  • Drug concentration ranges should span below and above the IC50 values for each compound

  • The ratio of drugs in combinations is typically fixed according to the ratio of their IC50 values

  • Both in vitro and in vivo models should be employed to validate findings

These approaches provide frameworks for exploring PPP2CA as a therapeutic target or biomarker for treatment response.

How can I address common technical challenges when working with PPP2CA antibodies?

When facing technical difficulties with PPP2CA detection:

• No signal or weak signal:

  • Ensure protein loading is sufficient (20-50 μg per lane for WB)

  • Increase antibody concentration (try 1:1000 instead of 1:4000)

  • Extend primary antibody incubation (overnight at 4°C)

  • Verify transfer efficiency with Ponceau S staining

  • Check positive control samples (e.g., HEK-293T, Jurkat, or MCF-7 cells)

• Multiple bands or non-specific binding:

  • Increase blocking time/concentration (5% BSA or milk for 1-2 hours)

  • Use more stringent washing (add 0.1% SDS to TBST)

  • Reduce primary antibody concentration

  • Try a different antibody targeting a different epitope

  • Consider recombinant monoclonal antibodies for higher specificity

• Inconsistent results:

  • Standardize lysate preparation (include phosphatase inhibitors)

  • Document antibody lot numbers (especially for polyclonal antibodies)

  • Use internal loading controls

  • Consider recombinant antibodies for better batch-to-batch consistency

• Application-specific issues:

  • For IHC: Test different antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • For IF: Optimize fixation and permeabilization conditions

  • For IP: Adjust antibody-to-lysate ratios within the recommended range (0.5-4.0 μg per 1.0-3.0 mg)

Methodical troubleshooting, along with appropriate controls, will help overcome most technical challenges.

How should I interpret conflicting data on PPP2CA function in different experimental systems?

When confronted with contradictory results:

• Consider context-dependent functions:

  • PPP2CA activity depends on its regulatory subunits, which vary across cell types

  • Compare PPP2CA expression levels across your experimental systems

  • Examine expression of other PP2A components (regulatory subunits)

  • Different stimuli may trigger distinct PPP2CA functions

• Methodological variations:

  • Document all experimental conditions meticulously

  • Consider timing differences (acute vs. chronic PPP2CA manipulation)

  • Validate findings with multiple techniques (genetic knockout, knockdown, inhibitors)

  • Verify the extent of PPP2CA manipulation in each system

• Substrate preferences:

  • Recent phosphoproteomic studies identified pSP/pTP motifs as predominant targets for PPP2CA

  • The prevalence of these motifs in your system may influence observable phenotypes

  • Analyze phosphorylation status of key substrates in your specific context

• Compensatory mechanisms:

  • Knockdown of PPP2CA may affect other PP2A components, as demonstrated in T cells where PPP2CA deficiency reduced PPP2CB, PPP2R5A, PPP2R5B, PPP2R1A, and PPP2R5C levels

  • These compensatory changes may differ between systems

  • Consider analyzing the entire PP2A network rather than PPP2CA in isolation

• Integration strategies:

  • Use systems biology approaches to reconcile disparate findings

  • Consider developing mathematical models that account for context-dependent effects

  • Collaborate with labs using different models to test predictions

Published literature shows context-dependent roles of PPP2CA in cancer and immune cells, highlighting the importance of system-specific validation.

What emerging technologies and approaches are advancing PPP2CA research?

Several cutting-edge approaches are transforming PPP2CA studies:

• Targeted protein degradation systems:

  • dTAG technology has been successfully applied to create dTAG/dTAG PPP2CA homozygous knock-in HEK293 cells

  • The PROTAC dTAG-13 enables selective and efficient degradation of endogenous dTAG-PPP2CA

  • This approach allows temporal control of PPP2CA levels without genetic compensation

• Advanced phosphoproteomics:

  • Tandem mass tag (TMT)-based quantitative analysis

  • Immobilized metal affinity chromatography-based phosphopeptide enrichment

  • These methods have revealed thousands of potential PPP2CA substrates

  • Identification of substrate motif preferences (e.g., pSP/pTP motifs)

• CRISPR/Cas9 applications:

  • Generation of PPP2CA knockout cell lines

  • Homozygous knock-in of tags for pulldown studies

  • Conditional knockout models in specific cell types (e.g., T cells)

• Multi-omics integration:

  • Combining phosphoproteomics with transcriptomics and proteomics

  • This approach revealed that PPP2CA deficiency affects not only phosphorylation but also protein expression levels of multiple PP2A components

  • Enables systems-level understanding of PPP2CA function

• Cytometric bead array:

  • Matched antibody pairs (e.g., 84155-2-PBS capture and 84155-4-PBS detection) allow quantitative detection of PPP2CA

  • Enables multiplexed analysis in complex samples

These technologies provide unprecedented insights into PPP2CA function and regulation, opening new avenues for therapeutic targeting and diagnostic applications.