Phospho-PIK3R2 (Y464) Antibody

What is PIK3R2 and why is the Y464 phosphorylation site significant?

PIK3R2, also known as phosphatidylinositol 3-kinase regulatory subunit beta or p85β, functions as a regulatory subunit of phosphoinositide-3-kinase (PI3K), a kinase that phosphorylates PtdIns(4,5)P2 to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3) . PIP3 plays a crucial role in recruiting PH domain-containing proteins to the membrane, including AKT1 and PDPK1, thereby activating signaling cascades involved in cell growth, survival, proliferation, motility, and morphology .

The Y464 phosphorylation site is particularly significant because:

• Phosphorylation at this site by FAK (Focal Adhesion Kinase) facilitates the nuclear translocation of p85β by enhancing its binding to KPNA1

• This phosphorylation event represents a regulatory mechanism that affects p85β's subcellular localization and function

• Nuclear p85β has been shown to perform oncogenic functions by repressing RB1 expression and regulating the G1/S cell cycle transition in certain cancers

What are the standard applications for Phospho-PIK3R2 (Y464) antibody in research?

Phospho-PIK3R2 (Y464) antibody is primarily used in the following research applications:

• Western Blot (WB): Used at dilutions ranging from 1:500 to 1:2000 to detect phosphorylated PIK3R2 at Y464 in cell and tissue lysates

• ELISA: Applied at dilutions of approximately 1:10000 for quantitative analysis of phosphorylated PIK3R2

• Dot Blot (DB): Used at dilutions of approximately 1:500

• Translocation studies: For investigating nuclear translocation of p85β and its interaction with other proteins like KPNA1

• Signal pathway analysis: For studying PI3K signaling in various physiological and pathological contexts

Each application requires specific optimization of antibody concentration, incubation conditions, and detection methods to achieve reliable results.

How should I optimize Western blot conditions for detecting phospho-PIK3R2 (Y464)?

Optimizing Western blot conditions for phospho-PIK3R2 (Y464) detection requires careful consideration of several parameters:

Sample preparation:

• Use fresh samples when possible

• Include phosphatase inhibitors in lysis buffers to preserve phosphorylation status

• Use reducing conditions as specified in protocols

• Load approximately 30-50μg of protein per lane

Electrophoresis conditions:

• Use 5-20% SDS-PAGE gels

• Run at 70V (stacking gel)/90V (resolving gel) for 2-3 hours

Transfer and detection:

• Transfer to nitrocellulose membrane at 150mA for 50-90 minutes

• Block with 5% non-fat milk/TBS for 1.5 hours at room temperature

• Incubate with primary antibody at 0.5-1μg/mL (or 1:500-1:2000 dilution) overnight at 4°C

• Wash with TBS-0.1% Tween three times (5 minutes each)

• Incubate with appropriate HRP-conjugated secondary antibody at 1:10000 dilution

• Develop using enhanced chemiluminescent detection

Expected results:

• The expected band size for phosphorylated PIK3R2 is approximately 85kDa

• Jurkat cell extracts can be used as positive controls

What controls should be included when studying phospho-PIK3R2 (Y464) in cell signaling experiments?

For rigorous validation of phospho-PIK3R2 (Y464) in cell signaling experiments, include the following controls:

• Positive controls:

  • Jurkat cell extracts

  • 293T whole cell lysates

  • Cells treated with growth factors or stimuli known to activate PI3K signaling

• Negative controls:

  • Samples treated with phosphatase to remove phosphorylation

  • Cells treated with PI3K inhibitors like LY294002

  • Samples from PIK3R2 knockdown/knockout cells

• Specificity controls:

  • Non-phosphorylated PIK3R2 antibody to compare total protein levels

  • Blocking peptide controls to confirm antibody specificity

  • Immunoprecipitation followed by Western blot with different antibodies

• Signal pathway validation:

  • Parallel detection of other PI3K pathway components (p110, AKT, etc.)

  • Use of FAK inhibitors like defactinib to modulate Y464 phosphorylation

  • Detection of downstream effectors to validate functional consequences

How can I differentiate between phosphorylation of PIK3R2 (Y464) and similar phosphorylation sites in PIK3R1 and PIK3R3?

Differentiating between phosphorylation of PIK3R2 (Y464) and similar sites in PIK3R1 (Y467) and PIK3R3 (Y199) requires careful experimental design:

• Antibody selection:

  • Use antibodies with validated specificity for each phosphorylation site

  • For PIK3R2-specific detection, select antibodies like those described in search results , , and

  • For detecting all three isoforms, use pan-specific antibodies like the Phospho-PIK3R1/PIK3R2/PIK3R3-Y467 Polyclonal Antibody (CABP0427)

• Validation approaches:

  • Perform immunoprecipitation with isoform-specific antibodies followed by Western blot with phospho-specific antibodies

  • Use recombinant proteins with single phosphorylation sites as standards

  • Employ siRNA/shRNA knockdown of specific isoforms to confirm signal specificity

• Advanced techniques:

  • Use mass spectrometry to distinguish phosphopeptides by their unique mass-to-charge ratios

  • Apply phosphoproteomics approaches to identify isoform-specific phosphorylation events

  • Employ high-content antibody microarrays for multiplexed detection and quantification

The observed molecular weights can also help differentiate the isoforms: PIK3R1 (p85α) at 85kDa, PIK3R2 (p85β) at 81-85kDa, and PIK3R3 (p55γ) at 55kDa .

What is the functional significance of nuclear translocation of phosphorylated PIK3R2, and how can it be studied?

Recent research has revealed that phosphorylation of p85β (PIK3R2) at Y464 by FAK facilitates its nuclear translocation, where it performs functions distinct from its cytoplasmic role in PI3K signaling .

Functional significance:

• Nuclear p85β performs oncogenic functions by repressing RB1 expression

• It regulates the G1/S cell cycle transition

• Nuclear p85β represses RB1 expression by stabilizing histone methyltransferase EZH1/EZH2 proteins

• High nuclear p85β expression is associated with clear cell renal cell carcinoma (ccRCC) tumorigenesis and patient survival

Methodologies for studying nuclear translocation:

• Subcellular fractionation:

  • Separate nuclear and cytoplasmic fractions using differential centrifugation

  • Validate fraction purity with markers (e.g., HDAC1 for nucleus, GAPDH for cytoplasm)

  • Measure phospho-PIK3R2 levels in each fraction by Western blot

• Immunofluorescence microscopy:

  • Use phospho-PIK3R2 (Y464) antibodies for immunostaining

  • Co-stain with nuclear markers (DAPI, lamin)

  • Quantify nuclear/cytoplasmic signal ratios

• Chemically inducible dimerization systems:

  • Apply rapamycin-dependent heterodimerization of FKBP and FRB domains

  • Use YFP-FKBP-iSH2 (YF-iSH2) and Lyn-CFP-FRB (Lyn-CR) constructs

  • Monitor translocation by live-cell imaging

• Mechanistic studies:

  • Investigate the interaction between phospho-PIK3R2 and nuclear importers like KPNA1

  • Use co-immunoprecipitation to detect protein-protein interactions

  • Apply FAK inhibitors like defactinib to modulate phosphorylation and nuclear translocation

How can I address non-specific binding when using phospho-PIK3R2 (Y464) antibodies?

Non-specific binding is a common challenge when working with phospho-specific antibodies. To address this issue:

• Optimize blocking conditions:

  • Test different blocking agents (BSA, non-fat milk, commercial blockers)

  • Increase blocking time (1.5-2 hours at room temperature)

  • Add 0.1-0.3% Tween-20 to wash buffers

• Antibody validation:

  • Use blocking peptides to confirm specificity

  • Test the antibody on samples with known phosphorylation status

  • Include phosphatase-treated samples as negative controls

• Dilution optimization:

  • Test a range of antibody dilutions (1:500 to 1:2000 for WB)

  • Reduce incubation time if background is high

  • Consider using more stringent wash conditions

• Sample preparation improvements:

  • Ensure complete protein denaturation

  • Use fresh samples with appropriate protease and phosphatase inhibitors

  • Consider immunoprecipitation to enrich for the target protein before Western blot

• Detection system optimization:

  • Use highly specific secondary antibodies

  • Consider fluorescent-based detection systems for better quantification

  • Apply enhanced chemiluminescent detection with appropriate exposure times

How do I interpret changes in phospho-PIK3R2 (Y464) levels in the context of PI3K pathway activity?

Interpreting changes in phospho-PIK3R2 (Y464) levels requires consideration of multiple factors:

• Relationship to PI3K catalytic activity:

  • Phosphorylation of PIK3R2 at Y464 can affect its interaction with the p110 catalytic subunit

  • Changes may not directly correlate with PIP3 production, as shown in studies with PI3K inhibitors like LY294002

  • The iSH2 domain of PIK3R2 can mediate endocytosis independently of PI3K catalytic activity

• Subcellular localization context:

  • Increased phosphorylation may indicate enhanced nuclear translocation rather than changes in cytoplasmic PI3K activity

  • Separate analysis of nuclear and cytoplasmic fractions is essential for comprehensive interpretation

  • Nuclear phospho-PIK3R2 may regulate gene expression independently of its role in PI3K signaling

• Temporal dynamics:

  • Consider the timing of measurements relative to stimulus application

  • Phosphorylation may be transient or sustained depending on the cellular context

  • Sequential activation of different components of the PI3K pathway may occur

• Multi-parameter analysis:

  • Measure phosphorylation of other PI3K pathway components (AKT, PDPK1) in parallel

  • Assess functional outcomes like cell proliferation, survival, or migration

  • Correlate phospho-PIK3R2 levels with expression of regulated genes like RB1

• Quantification approaches:

  • Normalize phospho-PIK3R2 levels to total PIK3R2 to account for expression changes

  • Use appropriate statistical analyses for multiple samples

  • Consider using phospho-antibody microarrays for higher-throughput analysis

How can high-content antibody microarrays be used to study phospho-PIK3R2 (Y464) in relation to other phosphoproteins?

High-content antibody microarrays offer powerful approaches for studying phospho-PIK3R2 (Y464) in the context of broader signaling networks:

• Methodology for microarray applications:

  • Microarrays can be prepared with hundreds of antibodies against different phosphoproteins

  • Samples labeled with fluorescent dyes are incubated on the arrays

  • Semi-quantitative measurements of multiple proteins can be obtained simultaneously

  • This approach requires only minute amounts of biological samples (biofluids, tissue extracts, cell lysates)

• Advantages for phospho-PIK3R2 research:

  • Allows simultaneous detection of multiple components in the PI3K pathway

  • Enables correlation of phospho-PIK3R2 (Y464) with other signaling events

  • Facilitates biomarker discovery in disease contexts

  • Permits tracking of temporal signaling dynamics across multiple pathways

• Implementation considerations:

  • Ensure inclusion of appropriate controls and normalization standards

  • Validate microarray findings with orthogonal methods like Western blot

  • Consider using statistical approaches like principal component analysis for data interpretation

  • Pay attention to sample preparation to preserve phosphorylation status

• Applications in disease research:

  • Can be used to identify dysregulated signaling networks in cancer samples

  • Enables biomarker discovery that may include phospho-PIK3R2 (Y464)

  • Allows for personalized medicine approaches based on phosphoprotein profiles

What are the implications of PIK3R2 (Y464) phosphorylation in cancer research, and how can the phospho-specific antibody contribute to this field?

Recent research has revealed significant implications of PIK3R2 (Y464) phosphorylation in cancer:

The phospho-PIK3R2 (Y464) antibody thus serves as a critical tool not only for basic research but also for translational cancer studies with potential clinical applications.