PIK3R6 Antibody

What is PIK3R6 and what biological pathways does it participate in?

PIK3R6 (Phosphoinositide 3-kinase regulatory subunit 6) functions as a crucial component of the PI3K/AKT signaling pathway. This regulatory subunit works in conjunction with catalytic subunits to form functional PI3K enzyme complexes involved in signal transduction. The PI3K/AKT pathway plays pivotal roles in regulating cellular growth, proliferation, survival, and metabolism across various cell types. Dysregulation of this pathway is commonly observed in cancer and several other pathological conditions, making PIK3R6 an important target for both basic research and therapeutic development .

What are the key applications for PIK3R6 antibody in research settings?

PIK3R6 antibodies serve multiple experimental applications essential for investigating PI3K signaling. The primary research applications include Western blotting (WB) for protein detection and quantification, enzyme-linked immunosorbent assay (ELISA) for sensitive protein measurement, immunohistochemistry (IHC) for tissue localization studies, and immunofluorescence (IF) for subcellular visualization. These techniques collectively enable researchers to examine PIK3R6 expression, localization, and functional interactions within experimental systems, providing crucial insights into PI3K signaling dynamics .

What are the optimal experimental conditions for using PIK3R6 antibody in Western blotting?

For optimal Western blotting results with PIK3R6 antibody, researchers should employ dilution ratios between 1:500 and 1:5000, with initial testing at 1:1000 recommended for polyclonal antibodies like PACO60997. Protein samples should be prepared with standard protocols incorporating protease inhibitors to prevent degradation. After SDS-PAGE separation and membrane transfer, blocking should be performed with 5% non-fat milk or BSA in TBST buffer. Overnight primary antibody incubation at 4°C typically yields optimal signal-to-noise ratios, followed by appropriate HRP-conjugated secondary antibody incubation and ECL-based detection. Researchers should validate the antibody using positive control samples containing PIK3R6 protein to confirm specificity .

How does PIK3R6 relate to other PI3K pathway components in cellular signaling?

PIK3R6 functions as one of several regulatory subunits in the PI3K family, working in concert with catalytic subunits to form functional PI3K enzyme complexes. Unlike the better-characterized p85α regulatory subunit (encoded by PIK3R1), PIK3R6 has distinct functions in specific cellular contexts. In PI3K signaling, these regulatory subunits control the localization, stability, and activity of the catalytic subunits, including p110δ. The regulatory subunits respond to receptor activation and facilitate downstream signaling through AKT, mTOR, and other effector molecules. This coordinated activity regulates critical cellular processes including cell growth, survival, and metabolism across different cell types, particularly in immune cells where PI3K signaling plays essential roles in development and function .

What methodological approaches should be employed to investigate PIK3R6's role in B cell development and antibody responses?

Investigating PIK3R6's role in B cell development requires multiple complementary approaches. Researchers should consider generating conditional knockout models (Cre-loxP system) targeting PIK3R6 in B cells at different developmental stages. Flow cytometry analysis examining markers like CD19, B220, IgM, and IgD will characterize developmental populations, while functional assays should assess proliferation (CFSE dilution), calcium flux, and apoptosis (Annexin V/PI staining). For antibody response assessment, immunize mice with T cell-dependent antigens (e.g., NP-CGG) and T cell-independent antigens to measure class-switching and affinity maturation through ELISA and ELISPOT. Single-cell RNA sequencing from various B cell populations will reveal transcriptional changes dependent on PIK3R6. In human samples, PIK3R6 antibody can detect expression in lymphoid tissues while phospho-specific antibodies against downstream targets (pAKT, pS6) can evaluate pathway activation. Class-switch recombination can be studied in vitro using isolated B cells stimulated with anti-CD40/IL-4 and monitoring immunoglobulin production by ELISA .

How can researchers distinguish between effects of PIK3R6 and other regulatory subunits (PIK3R1-5) when studying PI3K signaling?

Distinguishing between effects of different PI3K regulatory subunits requires multiple strategic approaches. Researchers should employ isoform-specific antibodies with validated specificity through immunoprecipitation followed by mass spectrometry or knockout controls. RNA interference utilizing carefully designed siRNAs or shRNAs with minimal off-target effects can selectively reduce expression of individual subunits. CRISPR/Cas9 gene editing creating clean knockouts of specific regulatory subunits provides definitive functional assessment. Isoform-selective inhibitors (when available) can be used in dose-response studies with demonstration of target engagement. Expression profiling across tissue types and developmental stages using qPCR and Western blotting helps identify contexts where certain subunits predominate. Co-immunoprecipitation studies determine specific binding partners of each regulatory subunit. Complementation experiments in knockout systems can assess the ability of one regulatory subunit to compensate for another. Finally, comparative phosphoproteomics after manipulation of different regulatory subunits can map downstream signaling networks unique to each isoform, potentially revealing PIK3R6-specific signaling branches .

What are the molecular mechanisms through which dysfunctional PIK3R6 contributes to disease pathogenesis?

Dysfunctional PIK3R6 can contribute to disease development through several molecular mechanisms. In cancer biology, altered PIK3R6 expression or activity may disrupt normal regulatory constraints on PI3K signaling, promoting sustained pathway activation that drives cell proliferation, survival, and metabolic reprogramming. Studies examining PIK3R6 in immune cells suggest potential roles in regulating Class Switch Recombination (CSR) and Somatic Hypermutation (SHM) in B cells, which are critical for proper antibody production. Disruption of these processes through altered PI3K signaling can contribute to immunodeficiency syndromes or autoimmunity. Additionally, PI3K pathway hyperactivation impacts FOXO transcription factors, potentially altering expression of genes controlling cell cycle, apoptosis, and metabolism. This can disrupt B cell development, impair negative selection of autoreactive B cells, and affect germinal center formation and antibody maturation. Understanding these mechanisms requires sophisticated experimental approaches including conditional genetic models, molecular analyses of signaling pathway components, and functional immunological assays .

What experimental controls are essential when examining PIK3R6 expression and function in tissue samples?

Rigorous experimental controls are critical when examining PIK3R6 in tissue samples. For antibody validation, researchers must include positive controls (tissues/cells known to express PIK3R6), negative controls (PIK3R6-knockout tissues or cells), and isotype controls (matched non-specific IgG) in parallel experiments. Technical controls for immunohistochemistry/immunofluorescence include secondary-antibody-only controls to assess non-specific binding and tissue autofluorescence evaluation for IF studies. Competitive blocking experiments using the immunizing peptide can verify antibody specificity. For expression analysis, researchers should analyze multiple reference genes (GAPDH, β-actin, HPRT) for normalization in qPCR and Western blotting. Functional studies require pathway inhibitor controls (pan-PI3K inhibitors, isoform-selective inhibitors) to confirm signaling connections. Tissue-specific considerations include accounting for cell type heterogeneity through co-staining with lineage markers in complex tissues. Finally, when examining pathological specimens, appropriate control tissues matched for age, gender, and tissue site are essential for accurate comparison and interpretation of results .

What are the recommended protocols for optimizing PIK3R6 antibody use in immunohistochemistry?

Optimizing PIK3R6 antibody use in immunohistochemistry requires careful attention to sample preparation and staining conditions. Researchers should first perform antigen retrieval optimization testing multiple methods (heat-induced epitope retrieval in citrate buffer pH 6.0, EDTA buffer pH 9.0, or enzymatic retrieval with proteinase K). Antibody titration experiments starting with recommended dilutions (1:200-1:500) and extending in both directions will determine optimal concentration. Blocking solutions containing 5-10% normal serum from the secondary antibody host species with 0.1-0.3% Triton X-100 for membrane permeabilization improve signal-to-noise ratio. Signal amplification systems (avidin-biotin complex, tyramide signal amplification) may enhance detection of low-abundance targets. Counterstaining with hematoxylin provides structural context without overwhelming the specific signal. Automated staining platforms can improve reproducibility for clinical applications. Positive controls (tissues known to express PIK3R6) and negative controls (primary antibody omission, isotype controls, pre-absorption with immunizing peptide) are essential for each staining run. Formalin-fixed paraffin-embedded tissues typically require 24-48 hour fixation times for optimal morphology while preserving antigenicity .

What are the key considerations for validating PIK3R6 antibody specificity in research applications?

Validating PIK3R6 antibody specificity requires a multi-faceted approach. Researchers should begin with Western blot analysis against recombinant PIK3R6 protein and lysates from cells expressing endogenous PIK3R6, confirming detection of appropriately sized bands. Genetic validation using PIK3R6 knockdown/knockout systems demonstrates antibody specificity through loss of signal. Peptide competition assays involve pre-incubating the antibody with excess immunizing peptide to block specific binding. Immunoprecipitation followed by mass spectrometry can identify proteins captured by the antibody, confirming PIK3R6 enrichment. Cross-reactivity testing against related PI3K regulatory subunits (PIK3R1-5) ensures isoform specificity. Reproducibility across multiple antibody lots should be established. Testing across multiple applications (WB, IHC, IF, ELISA) confirms consistent target recognition. Evaluation in multiple cell types/tissues known to express or lack PIK3R6 further validates specificity. Finally, correlation between protein detection and mRNA expression levels in the same samples provides additional validation of antibody performance .

How should researchers design experiments to study PIK3R6 interactions with signaling partners?

Designing experiments to study PIK3R6 protein interactions requires systematic approaches. Co-immunoprecipitation using PIK3R6 antibody followed by immunoblotting for suspected binding partners provides direct evidence of physical interactions. Reciprocal co-IP experiments pulling down known or suspected partners and probing for PIK3R6 confirm interactions from both perspectives. Proximity ligation assays (PLA) can visualize protein interactions in situ with subcellular resolution. FRET/BRET approaches using fluorescently tagged proteins allow real-time monitoring of interactions in living cells. For comprehensive interaction screening, immunoprecipitation coupled with mass spectrometry identifies the complete PIK3R6 interactome. Confirmation experiments should include appropriate controls like IgG negative controls, input samples, and tests for non-specific binding. Domain mapping through deletion or point mutation constructs can identify specific regions mediating interactions. Functional validation using overexpression or knockdown approaches determines biological significance of identified interactions. Stimulus-dependent interaction studies (before/after growth factor treatment or other stimuli) reveal dynamic regulation of the PIK3R6 interactome. Finally, computational analysis through pathway mapping software helps contextualize interactions within broader signaling networks .

What are the recommended approaches for quantifying PI3K pathway activation when studying PIK3R6 function?

Quantifying PI3K pathway activation requires multiple complementary approaches. Western blotting for phosphorylated downstream effectors including pAKT (Ser473 and Thr308), pS6K (Thr389), pS6 (Ser235/236), and pFOXO proteins provides direct evidence of pathway activity. Phospho-flow cytometry enables single-cell resolution analysis of pathway activation, particularly valuable for heterogeneous populations. ELISA-based phosphoprotein detection kits offer quantitative measurement with high sensitivity. Cellular assays measuring PIP3 production through PIP3-binding domain translocation or direct mass spectrometry quantification of phosphoinositides assess immediate pathway output. Transcriptional reporter assays using FOXO-responsive elements linked to luciferase can measure pathway activation through transcriptional readouts. Metabolic assays examining glucose uptake, glycolysis, and mitochondrial activity provide functional readouts of PI3K pathway effects. Inhibitor studies using validated PI3K pathway inhibitors at different levels (PI3K, AKT, mTOR) help establish causality in observed phenotypes. Time-course experiments capture dynamic pathway activation and deactivation kinetics. Finally, multiplexed approaches like reverse phase protein arrays or mass spectrometry-based phosphoproteomics provide comprehensive pathway activation profiles .

How can researchers address common technical challenges with PIK3R6 antibody applications?

Addressing technical challenges with PIK3R6 antibody applications requires systematic troubleshooting. For weak or absent Western blot signals, researchers should increase protein loading (50-100 μg), optimize antibody concentration, extend incubation time (overnight at 4°C), enhance detection methods (high-sensitivity ECL substrates), and verify sample preparation (check extraction buffers contain phosphatase/protease inhibitors). Background issues in immunostaining can be minimized by optimizing blocking conditions (try 5% BSA or normal serum from secondary antibody species), increasing washing steps (use 3-5 washes of 5-10 minutes each), and reducing primary/secondary antibody concentrations. Non-specific bands in Western blot can be addressed through more stringent washing conditions, using gradient gels for better resolution, and performing peptide competition controls. Batch-to-batch variability should be managed by purchasing larger antibody lots, thoroughly validating each new lot against previous lots, and maintaining consistent experimental protocols. For tissue-specific detection difficulties, researchers should optimize antigen retrieval methods (test heat-induced retrieval with different buffers and pH conditions), adjust fixation protocols (reduce fixation time), and test multiple antibody clones targeting different epitopes. These approaches systematically eliminate variables that may compromise experimental outcomes .

What are the potential pitfalls in data interpretation when studying PIK3R6 in disease models?

Data interpretation when studying PIK3R6 in disease models presents several challenges. Researchers must be cautious about attributing causality, as correlations between PIK3R6 expression changes and disease phenotypes require functional validation through gain/loss-of-function studies. Cell type heterogeneity in complex tissues can confound results, necessitating single-cell approaches or careful microdissection to isolate specific populations. Compensatory mechanisms involving other PI3K regulatory subunits may mask PIK3R6-specific effects, requiring comprehensive analysis of all related family members. Genetic background effects in animal models can significantly influence phenotypes, demanding the use of proper background-matched controls or backcrossing strategies. Translational relevance between model systems and human disease requires validation in multiple models and human samples. Disease stage-dependent effects mean PIK3R6's role may differ during initiation versus progression phases. Off-target effects of pharmacological tools necessitate genetic validation approaches. Context-dependent signaling where PIK3R6's function varies by cell type and stimulus requires testing multiple conditions. Finally, researchers should consider post-translational modifications of PIK3R6 that may not be detected by standard antibodies but significantly affect function .

How should researchers resolve conflicting results between different experimental approaches when studying PIK3R6?

Resolving conflicting results in PIK3R6 research requires systematic investigation. First, researchers should examine methodological differences through detailed protocol comparison and standardization of critical parameters like antibody concentrations, incubation times, and detection methods. Technical validation using multiple antibodies targeting different PIK3R6 epitopes helps confirm findings are not antibody-specific artifacts. Biological validation across different cell lines or primary cells addresses cell type-specific effects. Assay sensitivity differences should be considered, as some techniques (mass spectrometry, ELISA) may detect lower expression levels than others (Western blot, IHC). Temporal dynamics may explain discrepancies if different approaches capture distinct time points in dynamic processes. Sample preparation variations in protein extraction methods, fixation protocols, or buffer compositions can significantly impact results. When genetic approaches (CRISPR, siRNA) and pharmacological approaches (inhibitors) yield different outcomes, researchers should evaluate specificity and completeness of target engagement. For conflicting in vitro versus in vivo results, researchers should consider microenvironment contributions and systemic effects. Finally, when literature conflicts with current findings, direct replication of published methods may identify sources of variation. These approaches systematically identify variables contributing to experimental discrepancies .

What emerging technologies might enhance our understanding of PIK3R6 function in normal and pathological contexts?

Emerging technologies offer powerful new approaches for investigating PIK3R6 biology. CRISPR-based gene editing enables precise manipulation of PIK3R6 genomic elements, including regulatory regions and specific domains, providing insights into structure-function relationships. CRISPRi/CRISPRa systems allow temporal control of gene expression without permanent modification. Single-cell technologies including scRNA-seq, scATAC-seq, and spatial transcriptomics reveal cell-type-specific expression patterns and regulatory mechanisms across tissues and disease states. Organoid models recapitulating complex tissue architecture provide physiologically relevant systems for studying PIK3R6 function in human cells. Advanced imaging techniques like super-resolution microscopy and live-cell imaging visualize PIK3R6 localization and dynamics at unprecedented resolution. Proximity labeling approaches (BioID, APEX) identify protein interaction networks in native cellular contexts. Phosphoproteomics coupled with kinase activity profiling characterizes signaling networks downstream of PIK3R6. Patient-derived xenograft models enable investigation of PIK3R6 in human disease contexts within in vivo environments. High-throughput drug screening platforms facilitate identification of compounds modulating PIK3R6-dependent signaling. These technologies collectively promise deeper understanding of PIK3R6's physiological roles and potential therapeutic targeting strategies .

How might understanding PIK3R6 function contribute to development of targeted therapies for diseases involving PI3K pathway dysregulation?

Understanding PIK3R6 function could significantly impact therapeutic development for PI3K-related diseases. Detailed characterization of PIK3R6's structural interactions with catalytic subunits may reveal unique binding interfaces for selective drug targeting that avoids the toxicity associated with pan-PI3K inhibition. Identification of PIK3R6-specific effector pathways could uncover novel downstream targets for therapeutic intervention that maintain efficacy while reducing off-target effects. Tissue and cell-type specific expression patterns of PIK3R6 might enable development of targeted delivery strategies for pathway inhibitors to affected tissues. Studies of PIK3R6 in immune regulation could inform immunotherapy approaches, particularly for conditions involving B cell dysfunction where PI3K signaling plays crucial roles in development and activation. Biomarker development based on PIK3R6 expression, mutation status, or activity could enable patient stratification for clinical trials and personalized treatment selection. Combination therapy strategies might be developed by understanding synergistic relationships between PIK3R6-dependent signaling and other oncogenic or inflammatory pathways. Drug resistance mechanisms related to PIK3R6 compensation or adaptation could inform sequential or alternating treatment strategies. Finally, understanding PIK3R6's role in normal physiology helps predict and mitigate potential adverse effects of therapeutic targeting .

What approaches should researchers use to investigate the role of PIK3R6 in immunological disorders and cancer?

Investigating PIK3R6 in immunological disorders and cancer requires comprehensive approaches spanning molecular, cellular, and in vivo studies. Researchers should conduct genetic association studies analyzing PIK3R6 variants in patient cohorts with immunodeficiencies, autoimmune conditions, and cancer subtypes. Expression profiling across immune cell subsets and cancer tissues using qPCR, immunoblotting, and single-cell RNA sequencing identifies contexts where PIK3R6 is dynamically regulated. Functional studies in primary immune cells should examine effects of PIK3R6 modulation on activation, differentiation, cytokine production, and effector functions. For cancer research, transformation assays, proliferation, migration, invasion, and anchorage-independent growth studies reveal oncogenic potential. Conditional knockout mouse models with tissue-specific or inducible PIK3R6 deletion enable in vivo assessment of immune development, responses to pathogens, autoimmune tendency, and tumor susceptibility. Mechanistic investigations should characterize PIK3R6 protein interactions, post-translational modifications, and subcellular localization in normal versus disease states. Pharmacological studies using pathway inhibitors in patient-derived samples can assess therapeutic potential and predictive biomarkers. Finally, translational validation in human biospecimens correlating PIK3R6 alterations with clinical outcomes establishes relevance to human disease .

What computational and bioinformatic approaches can enhance PIK3R6 research?

Computational and bioinformatic approaches substantially enhance PIK3R6 research across multiple dimensions. Structural modeling using homology modeling and molecular dynamics simulations predicts PIK3R6 protein structure, binding interfaces, and potential drug interaction sites. Network analysis integrating protein-protein interaction data, gene co-expression patterns, and pathway databases contextualizes PIK3R6 within broader signaling networks. Multi-omics data integration combining genomics, transcriptomics, proteomics, and phosphoproteomics datasets reveals regulatory relationships and functional impacts of PIK3R6 alterations. Machine learning approaches analyzing large-scale cancer genomics databases can identify patterns associating PIK3R6 alterations with disease subtypes and clinical outcomes. Evolutionary analysis comparing PIK3R6 across species provides insights into conserved functional domains and species-specific adaptations. Variant effect prediction tools assess functional implications of PIK3R6 mutations identified in patient samples. Gene set enrichment analysis and pathway analysis of PIK3R6-associated genes identifies biological processes and functions linked to PIK3R6 activity. Systems biology modeling through ordinary differential equations or Boolean networks simulates dynamic PIK3R6 signaling behaviors. Drug repurposing algorithms can identify existing compounds that might modulate PIK3R6-dependent pathways. These computational approaches complement experimental studies by generating testable hypotheses and providing interpretive frameworks for complex datasets .