PPP4C Antibody

What is PPP4C and what are its primary cellular functions?

PPP4C is the catalytic subunit of the PP4 serine/threonine protein phosphatase. It functions as part of a holoenzyme comprising the PPP4C catalytic subunit in association with regulatory subunits (typically PP4-R1 or PP4-R2). PPP4C is involved in multiple cellular processes including:

• Direct interaction with and activation of NF-kappaB

• Interaction with histone deacetylase HDAC3 to influence transcription

• Microtubule organization during mitosis

• Regulation of MAPK and JNK-1 signaling pathways

• Maturation of spliceosomal snRNPs

• Apoptosis and DNA repair mechanisms

• DNA damage checkpoint signaling

For effective study of these functions, researchers should consider using PPP4C antibodies in conjunction with functional assays that directly measure the phosphatase activity, such as the DiFMUP substrate assay which can be used with immunoprecipitated PPP4C .

Both polyclonal and monoclonal antibodies against PPP4C are available for research use, each with distinct characteristics:

Polyclonal PPP4C antibodies:

• Typically produced in rabbits

• Recognize multiple epitopes on the PPP4C protein

• Generally offer higher sensitivity but potentially lower specificity

• Particularly useful for applications like western blotting and immunoprecipitation where signal amplification is beneficial

• Example: Rabbit anti-PPP4C antibodies that are affinity purified against specific PPP4C epitopes

Monoclonal PPP4C antibodies:

• Available as mouse monoclonal antibodies

• Recognize a single epitope on the PPP4C protein

• Provide higher specificity and consistency between batches

• Particularly valuable for distinguishing between closely related proteins

• Example: Mouse monoclonal antibody clone 2F11-D10-G4 which specifically detects endogenous levels of PPP4C without cross-reactivity to related proteins

When choosing between these antibody types, consider the specificity requirements of your experiment and the nature of your biological question.

How can PPP4C antibodies be utilized in cancer research?

PPP4C has emerged as an important target in cancer research, particularly in lung adenocarcinoma (LUAD) and ovarian cancer studies. Methodological approaches include:

For expression analysis:

• Immunohistochemical staining to validate PPP4C overexpression in tumor tissues compared to normal tissues

• Western blotting to quantify expression across cancer cell line panels

For mechanistic studies:

• Combined PPP4C knockdown with immunoblotting to assess effects on signaling pathways

• Correlation of PPP4C expression with immune cell infiltration using algorithms like CIBERSORT

• Construction of prognostic models incorporating PPP4C expression data

Recent research has shown that PPP4C is significantly overexpressed in ovarian cancer compared to normal ovarian and fallopian tube tissues, while expression of regulatory subunits varies. Interestingly, at the protein level, PPP4C is robustly expressed across high-grade serous ovarian cancer (HGSOC) cell lines, with variable expression of regulatory subunits like PPP4R3β .

What methodologies can be employed to study PPP4C's role in immune responses?

PPP4C plays critical roles in immune function, particularly in T cell proliferation and adaptive immune responses. Research approaches include:

For T cell studies:

• Using PPP4C antibodies to track expression during T cell activation

• Combining with qPCR to validate genomic deletion efficiency of ppp4c gene in conditional knockout models

• Analysis of phosphorylation status of downstream targets through western blotting

For NK cell function:

• Functional assessment of PP4 knockdown on NK cell activation and cytolytic activity

• Immunoprecipitation of PPP4C followed by phosphatase activity assays using substrates like DiFMUP

• Analysis of immunological profiles using the ESTIMATE method and Spearman's rank correlation

In ovarian cancer research, PP4 inhibition has been shown to sensitize cancer cells to NK cell-mediated killing, representing a potential therapeutic strategy .

How can bioinformatic approaches be integrated with antibody-based detection of PPP4C?

Modern PPP4C research often combines traditional antibody techniques with bioinformatic analysis:

Integrated methodological approaches:

• Correlating protein expression levels (detected by antibodies) with RNA-seq data

• Using The Tumor IMmune Estimation Resource (TIMER) to analyze correlation between PPP4C expression and immune cell infiltration

• Leveraging databases like DepMap to obtain RNA level expression values and proteomic scores for PPP4C across cell lines

• Constructing prognostic models using PPP4C expression data combined with immune-related gene profiles

For comprehensive analysis, researchers examining PPP4C should consider analyzing all PP4 complex members (PPP4C, PPP4R3A, PPP4R3B, and PPP4R2) across different cancer types using resources like the pan-cancer TCGA data available at cBioPortal, UCSC Cancer Browser, and DepMap database .

What are optimal protocols for PPP4C immunoprecipitation?

For successful immunoprecipitation of PPP4C, the following methodology has been validated:

• Prepare cell/tissue lysates (typically 1 mg total protein)

• Incubate with anti-PPP4C antibody (typically rabbit polyclonal) or control IgG

• Add Protein-A Agarose beads and incubate overnight at 4°C

• Wash beads thoroughly with appropriate buffer

• For activity assays: Resuspend beads in assay buffer (30 mM HEPES, 0.1 mg/mL BSA, 0.1 mM MnCl₂, 1 mM sodium ascorbate, 1 mM DTT, 0.01% Triton X-100)

• For inhibition studies: Include fostriecin (1 nM) during incubation

• For phosphatase activity measurement: Add DiFMUP substrate (100 μM) and measure fluorescence at 450 nm after 60 minutes

This approach allows both detection of PPP4C protein interactions and functional assessment of its phosphatase activity in a single experiment.

When implementing PPP4C antibodies in new systems, comprehensive validation is essential:

Recommended validation approaches:

• Knockout/knockdown controls: Validate specificity using siRNA/shRNA knockdown or CRISPR knockout of PPP4C

  • Human and mouse siGENOME SMARTpool PPP4C siRNA can be used with Lipofectamine 3000

  • For stable knockdown, lentiviral vectors like pLKO.1 with PPP4C shRNA followed by puromycin selection are effective

• Expression validation across multiple techniques:

  • Compare protein detection with mRNA expression (RT-qPCR)

  • Validate across multiple applications (WB, IHC, IF) to ensure consistent results

• Orthogonal validation:

  • Compare results with RNAseq data where available

  • Use multiple antibodies targeting different epitopes of PPP4C

• Positive control samples:

  • HEK-293 cells, Jurkat cells, and mouse kidney tissue have been validated as positive controls for PPP4C expression

How can researchers address non-specific binding when using PPP4C antibodies?

Non-specific binding can compromise experimental results when working with PPP4C antibodies:

Methodological solutions:

• Optimization of blocking conditions:

  • Use 3-5% BSA in PBS or TBS for western blotting applications

  • Consider adding 0.1-0.3% Triton X-100 for permeabilization in IF/ICC applications

• Antibody dilution optimization:

  • For western blotting: Test dilutions between 1:500-1:2000

  • For immunofluorescence: Begin with 1:200-1:800 dilutions

• Sample preparation considerations:

  • For tissue sections: Optimize antigen retrieval methods (heat vs. enzymatic)

  • For cell lines: Test different fixation methods (paraformaldehyde vs. methanol)

• Controls to implement:

  • Include isotype control antibodies (rabbit or mouse IgG depending on antibody host)

  • Run parallel experiments with PPP4C knockdown/knockout samples

How can PPP4C antibodies be used to distinguish between different PP4 complexes?

PPP4C functions as part of different holoenzyme complexes with distinct regulatory subunits and functions:

Methodological approach for complex identification:

• Co-immunoprecipitation strategy:

  • Immunoprecipitate with anti-PPP4C antibody

  • Probe western blots with antibodies against specific regulatory subunits (PPP4R1, PPP4R2, PPP4R3A, PPP4R3B)

  • This allows identification of which regulatory subunits are associated with PPP4C in your specific context

• Functional assessment:

  • The PPP4C-PPP4R1 complex may regulate HDAC3

  • The PPP4C-PPP4R2-PPP4R3A complex specifically dephosphorylates H2AX

  • Assess these specific substrates to infer which complex is active

• Expression correlation analysis:

  • Analyze correlation between PPP4C and its regulatory subunits

  • In ovarian cancer, for example, variable expression of PPP4R3β has been observed across cell lines

What considerations are important when using PPP4C antibodies in cancer tissue microarrays?

Cancer tissue analysis presents unique challenges for PPP4C antibody applications:

Methodological considerations:

• Tissue heterogeneity management:

  • Consider using laser capture microdissection to isolate specific cell populations

  • Compare PPP4C staining patterns between tumor and adjacent normal tissues

  • Analyze correlation with markers of different cell types (epithelial, stromal, immune)

• Quantification approaches:

  • Implement digital pathology scoring systems for objective assessment

  • Consider H-score method (intensity × percentage positive cells)

  • Use image analysis software for automated quantification

• Correlation with clinical parameters:

  • PPP4C expression has been linked to poor prognosis in lung adenocarcinoma

  • Elevated expression correlates with immune cell infiltration patterns

  • Risk assessment models incorporating PPP4C and immune-related genes have shown prognostic value

• Complementary methodologies:

  • Combine IHC with RNA-seq data from the same tumor samples

  • Use multiplexed immunofluorescence to co-localize PPP4C with its interacting partners

  • Validate findings across multiple patient cohorts