PPP2R2B Antibody

What is PPP2R2B and why is it significant in scientific research?

PPP2R2B (also known as B55β) is a regulatory subunit of protein phosphatase 2A (PP2A), one of the major serine/threonine phosphatases involved in the negative control of cell growth and division. PPP2R2B modulates substrate selectivity and catalytic activity of PP2A while also directing the localization of the catalytic enzyme to specific subcellular compartments .

This protein is particularly significant in research because:

• It regulates the PI3K/AKT pathway, linking it to cell proliferation and survival through interactions with proteins like AKT1 and mTOR

• It is critical for normal brain function and neuronal survival through regulation of mitochondrial fission and fusion balance

• It plays a key role in immune response termination, with altered expression contributing to autoimmune pathology

• Mutations in PPP2R2B are associated with neurodevelopmental disorders and spinocerebellar ataxia 12 (SCA12)

What are the structural characteristics of PPP2R2B protein that researchers should be aware of?

PPP2R2B is a 52 kDa protein (443 amino acids) that functions as part of the heterotrimeric PP2A holoenzyme complex. Key structural features include:

• A relatively large CpG island encompassing its first exon, 5' UTR, and parts of the first intron, which is subject to methylation regulation

• Multiple isoforms generated through differential promoter usage and alternative splicing

• Specific interfaces for interaction with PP2A A and C subunits to form the functional holoenzyme

• A CAG trinucleotide repeat sequence (normally 7-28 copies) in the 5' UTR of some variants, which can be expanded to 66-78 copies in SCA12 cases

• Distinct domains that mediate its localization to mitochondria in certain isoforms (particularly isoform 2)

How should researchers optimize Western blot protocols when using PPP2R2B antibodies?

For optimal Western blot results with PPP2R2B antibodies:

• Sample preparation:

  • Use Triton X-100 soluble cell lysates for effective protein extraction

  • Heat samples at 95°C and load equal protein amounts onto 10% acrylamide gels

• Electrophoresis and transfer:

  • Separate proteins at constant voltage of 180V for approximately 45 minutes

  • Transfer to nitrocellulose membrane at constant current of 1 Amp for 2 hours

• Blocking and antibody incubation:

  • Block membranes in 2% bovine serum albumin supplemented with 0.05% sodium azide (30 min at 37°C)

  • Wash with TTBS (150 mM NaCl, 10 mM Tris pH 7.5, 0.01% Triton X-100)

  • Incubate with primary PPP2R2B antibody overnight at 4°C using recommended dilutions:

    • For monoclonal antibodies: 1:2000 dilution

    • For polyclonal antibodies: 1:500-1:6000 range, depending on specific antibody

  • Use fluorescent dye-conjugated secondary antibodies (1:15000 dilution) for 1 hour at room temperature

• Detection:

  • The expected band size for PPP2R2B is 52 kDa

  • Include appropriate positive controls (mouse brain tissue, SH-SY5Y cells, or HepG2 cells show reliable expression)

What are the most reliable tissue samples and cell lines for validating PPP2R2B antibody specificity?

Based on published research, the following samples provide reliable expression for PPP2R2B antibody validation:

Researchers should note that PPP2R2B is differentially expressed across tissues, with brain tissue showing the highest expression levels, making it the ideal positive control for antibody validation .

How can researchers distinguish between different isoforms of PPP2R2B in experimental settings?

Distinguishing between PPP2R2B isoforms requires careful experimental design:

• Antibody selection:

  • Use isoform-specific antibodies when available. For example, antibody VI-E6-2C6 specifically recognizes Bβ2 but not Bβ1 isoform

  • Verify epitope locations - antibodies targeting the N-terminal region may distinguish between isoforms with different N-termini

• Molecular weight discrimination:

  • Different isoforms may show slight variations from the expected 52 kDa size

  • Use high-resolution SDS-PAGE (10-12%) with extended run times for better separation

• RT-PCR validation:

  • Design isoform-specific primers targeting unique exons to confirm expression at mRNA level

  • Quantitative RT-PCR can validate antibody results by measuring relative isoform abundance

• Recombinant expression systems:

  • Use tagged recombinant isoforms as positive controls for size verification

  • The NanoBiT split luciferase complementation assay has been used successfully to investigate specific PPP2R2B isoform incorporation into PP2A holoenzymes

• Immunoprecipitation followed by mass spectrometry:

  • This can definitively identify specific isoforms based on unique peptide sequences

What are common sources of false positives/negatives when using PPP2R2B antibodies, and how can researchers avoid them?

Common sources of false results and solutions:

Technical validation approaches:

• Confirm antibody specificity using overexpression or knockdown/knockout models

• Validate results with at least two different antibodies recognizing distinct epitopes

• Include positive control samples (brain tissue) in all experiments

How can PPP2R2B antibodies be utilized to investigate epigenetic regulation in autoimmune diseases?

PPP2R2B undergoes epigenetic regulation through CpG methylation that affects its expression, particularly in autoimmune diseases. Research approaches using PPP2R2B antibodies include:

• Combined ChIP-Western analysis:

  • Use chromatin immunoprecipitation with antibodies against methylation markers (e.g., 5-methylcytosine)

  • Follow with Western blotting using PPP2R2B antibodies to correlate methylation with protein expression

  • This approach revealed that systemic inflammation drives hypermethylation of PPP2R2B in autoimmune diseases

• Cell-specific expression profiling:

  • Use PPP2R2B antibodies in combination with cell-type markers to identify which immune cells show altered PPP2R2B expression

  • Apply in flow cytometry or immunofluorescence microscopy to correlate with disease activity

• Signaling pathway analysis:

  • Investigate PPP2R2B's role in resistance to cytokine withdrawal-induced death (CWID) in T cells

  • Use PPP2R2B antibodies to track protein levels before and after IL-2 withdrawal in healthy vs. autoimmune patient T cells

  • Findings show that PPP2R2B expression increases ~3-fold after IL-2 withdrawal in healthy donors but remains low in SLE patients

• TNF-α induction models:

  • Use PPP2R2B antibodies to monitor protein changes in healthy T cells exposed to TNF-α

  • This can replicate the phenotype observed in autoimmune diseases and help establish causality between inflammation and PPP2R2B suppression

What methodologies can researchers employ to study PPP2R2B involvement in neurodevelopmental disorders using available antibodies?

Recent research has identified de novo missense variants in PPP2R2B as causes of neurodevelopmental syndromes. Advanced methodological approaches include:

• PP2A holoenzyme assembly analysis:

  • Use split luciferase complementation assays with PPP2R2B antibodies to assess mutant protein incorporation into the PP2A complex

  • Co-immunoprecipitation with FLAG-tagged PPP2R2B followed by Western blotting can quantify binding to endogenous A and C subunits

• Protein turnover assessment:

  • Apply pulse-chase experiments with HaloTag fusion proteins to measure PPP2R2B stability

  • Western blotting with PPP2R2B antibodies can normalize fluorescence signals to total protein levels

  • This revealed that neurodevelopmental disorder-associated variants accelerate PPP2R2B turnover

• Phosphatase activity measurement:

  • Isolate PPP2R2B-GFP complexes using anti-GFP nanobodies

  • Conduct in vitro phosphatase assays using phospho-threonine peptide substrates

  • Correlate activity with Western blot detection of catalytic subunit levels in immunoprecipitates

• Mitochondrial dynamics investigation:

  • Use immunofluorescence with PPP2R2B antibodies to track mutant protein localization to mitochondria

  • Assess effects on mitochondrial fission/fusion and dephosphorylation of dynamin-related protein 1

  • This connects PPP2R2B variants to altered mitochondrial dynamics in neurodevelopmental disorders

How should researchers interpret differences in PPP2R2B expression levels across diverse experimental models?

Interpreting PPP2R2B expression data requires consideration of several factors:

• Tissue-specific expression patterns:

  • PPP2R2B shows highest expression in brain tissue with variable expression in other tissues

  • When comparing across tissues, normalize to tissue-specific positive controls rather than making direct comparisons

• Isoform-specific expression:

  • Different promoters and alternative splicing generate multiple isoforms

  • Ensure antibodies detect the specific isoforms of interest for your study

  • Consider using RT-PCR with isoform-specific primers to complement protein data

• Developmental and disease-specific regulation:

  • PPP2R2B expression changes during development and in disease states

  • In autoimmune conditions, PPP2R2B shows heterogeneous response patterns

  • Classify samples into response groups (normal induction, no change, decrease) rather than relying solely on mean values

• Epigenetic influences:

  • CpG methylation significantly impacts PPP2R2B expression

  • Consider measuring methylation status alongside protein levels, especially in inflammatory conditions

  • Unsupervised hierarchical clustering based on cytosine methylation can reveal distinct patient subgroups

• Standardized quantification approach:

  • Use density ratio comparisons to housekeeping proteins

  • Report fold-changes relative to appropriate controls

  • Consider presenting individual data points alongside group means to show sample heterogeneity

What are the critical factors to consider when analyzing PPP2R2B phosphatase activity in relation to protein expression?

When studying PPP2R2B's functional activity in relation to its expression levels, researchers should consider:

• Holoenzyme assembly vs. monomeric protein:

  • PPP2R2B protein detection may not correlate with functional PP2A activity

  • Monomeric PPP2R2B is rapidly degraded by the ubiquitin-proteasome pathway

  • Assess both total protein levels and incorporation into PP2A holoenzyme

• Activity measurement standardization:

  • Normalize phosphatase activity to the amount of catalytic subunit in the complex

  • Use colorimetric assays with molybdate/malachite green-based detection

  • Plot activity over time (0-45 min) rather than single endpoints

• Substrate specificity considerations:

  • Different substrates may show variable dependence on PPP2R2B

  • Standard substrates include phospho-threonine peptides like RRA(pT)VA

  • Consider testing physiological substrates relevant to your research question

• Competing regulatory mechanisms:

  • Post-translational modifications of PPP2R2B can affect activity without changing expression

  • Protein-protein interactions may sequester PPP2R2B without affecting detection

  • Consider analyzing PPP2R2B in different subcellular fractions

• Models for pathogenic variant assessment:

  • Compare wild-type vs. variant PPP2R2B effects on phosphatase activity

  • Include established non-functional mutants (e.g., RR168EE) as negative controls

  • Establish dose-response relationships rather than single-concentration comparisons