PIK3C3 Antibody, FITC conjugated

What is PIK3C3 and why is it an important research target?

PIK3C3, also known as VPS34, is the catalytic subunit of the sole class III phosphatidylinositol-3-kinase that specifically phosphorylates phosphatidylinositol to generate phosphatidylinositol-3-phosphate (PtdIns3P) . This enzyme plays a critical role in autophagy, vesicular trafficking, and cell signaling processes. PIK3C3 forms several functional complexes with regulatory proteins including PIK3R4/VPS15 and BECN1/Beclin-1, which determine its subcellular localization and specific functions . The importance of PIK3C3 in cellular homeostasis makes it a significant target for studying cellular responses to nutrient stress, membrane dynamics, and disease mechanisms related to autophagy dysregulation .

What is the purpose of FITC conjugation in PIK3C3 antibodies?

FITC conjugation provides direct fluorescent visualization of the PIK3C3 protein without requiring secondary antibody detection steps. The FITC fluorophore emits green fluorescence (peak emission ~520 nm) when excited at ~495 nm, making it compatible with standard fluorescence microscopy filter sets . This conjugation allows for:

• Direct visualization in immunofluorescence microscopy

• Flow cytometry analysis without secondary antibodies

• Multiplex staining when combined with antibodies conjugated to spectrally distinct fluorophores

• Reduced background in experiments where cross-reactivity with secondary antibodies is problematic

For example, in studies examining PIK3C3 localization during autophagy induction, FITC-conjugated antibodies provide immediate visualization of protein distribution changes without additional staining steps .

What are the recommended protocols for immunofluorescence using PIK3C3 Antibody, FITC conjugated?

For optimal immunofluorescence results with FITC-conjugated PIK3C3 antibodies, the following methodological approach is recommended:

• Fix cells with 4% formaldehyde for 10 minutes at room temperature

• Permeabilize with buffer containing 0.5% Triton X-100 for 30 minutes

• Block with 5% BSA in PBS for 30-60 minutes

• Incubate with FITC-conjugated PIK3C3 antibody (typically at 1:200-1:400 dilution) for 1-2 hours at room temperature or overnight at 4°C

• Wash extensively with PBS (3-5 times, 5 minutes each)

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

For tissue sections, additional steps include:

• Dewaxing with xylene for paraffin sections

• Antigen retrieval (typically heat-induced in citrate buffer)

• Longer primary antibody incubation (overnight at 4°C is standard)

• More extensive washing to reduce background

Visualization should be performed using appropriate filters for FITC (excitation ~495 nm, emission ~520 nm) using fluorescence or confocal microscopy .

How can PIK3C3 Antibody, FITC conjugated be used to study autophagy regulation?

PIK3C3 antibodies are valuable tools for investigating autophagy regulation at multiple levels:

Autophagosome Formation Dynamics:
FITC-conjugated PIK3C3 antibodies enable direct visualization of PIK3C3 recruitment to pre-autophagosomal structures. Researchers can induce autophagy through nutrient deprivation (using EBSS medium) and monitor temporal changes in PIK3C3 localization . This approach has revealed that PIK3C3 complex I (containing ATG14) localizes to autophagosome formation sites within minutes of starvation stimulus .

Differential Complex Formation:
PIK3C3 forms distinct complexes (PtdIns3K-C1 with ATG14 and PtdIns3K-C2 with UVRAG) that serve different functions in autophagy. Using co-immunofluorescence with markers for these complex components alongside FITC-PIK3C3 antibodies allows researchers to distinguish which complex is active under specific conditions .

Experimental Protocol for Autophagy Studies:

• Seed cells in appropriate culture vessels

• Induce autophagy through:

  • Nutrient starvation (EBSS medium for 2-4 hours)

  • mTOR inhibition (rapamycin treatment)

  • ER stress induction

• Fix and process for immunofluorescence

• Co-stain with FITC-PIK3C3 antibody and markers for:

  • Autophagosome formation (LC3B)

  • Lysosomal markers (LAMP-1)

• Analyze colocalization using confocal microscopy

The study by Liu et al. demonstrated that in PIK3C3 transgenic pig fibroblasts, accumulated LC3II protein was cleared faster than in wild-type cells during EBSS-induced autophagy, indicating enhanced autophagic flux .

What experimental controls should be included when using PIK3C3 Antibody, FITC conjugated in research?

For rigorous scientific investigation using FITC-conjugated PIK3C3 antibodies, the following controls are essential:

Antibody Specificity Controls:

• Isotype control: Use FITC-conjugated non-specific IgG from the same host species

• Antigen competition: Pre-incubate the antibody with excess immunogenic peptide to block specific binding

• Genetic validation: Use PIK3C3 knockout or knockdown cell lines to verify specific staining

Technical Controls:

• Autofluorescence control: Examine unstained samples to identify and subtract natural cellular fluorescence

• Secondary antibody-only control (if using amplification techniques)

• Single-color controls in multiplex experiments to set proper compensation

Biological Controls:

• Positive control: Include samples with known PIK3C3 upregulation (e.g., cells under starvation)

• Negative control: Include samples with PIK3C3 inhibition (e.g., wortmannin treatment)

• Time-course controls when studying dynamic processes

Quantitative Controls:

• Standardized reference samples for flow cytometry

• Fluorescence intensity calibration standards for microscopy

• Internal control proteins for normalization in Western blot applications

How does PIK3C3 function in the MTOR signaling pathway during nutrient stress?

PIK3C3 serves as a critical integration point between nutrient sensing and autophagy induction through its interaction with the MTOR (Mechanistic Target of Rapamycin) pathway:

Mechanism of Regulation:
Under nutrient-rich conditions, active MTOR complex 1 (MTORC1) phosphorylates and inhibits the ULK1 complex, preventing autophagy initiation. During nutrient starvation, MTORC1 activity decreases, relieving this inhibition. Simultaneously, PIK3C3 activity increases, generating PtdIns3P at autophagosome formation sites .

Research has demonstrated that MTORC1, but not MTORC2, directly regulates PIK3C3 activity. This was confirmed through experiments with Rptor (Raptor) knockdown cells, which showed minimal response to nutrient conditions, while Rictor knockout cells maintained normal responses similar to wild-type cells .

Experimental Evidence:
Studies visualizing PtdIns3P production (using GST-2XFYVE domain fusion protein) have shown that nutrient starvation rapidly increases PIK3C3-dependent PtdIns3P generation. This spatial and temporal regulation of PIK3C3 activity provides the biochemical foundation for autophagosome biogenesis .

The functional connection between MTOR and PIK3C3 creates a mechanistic link between amino acid starvation and autophagy induction via the direct activation of the autophagy-specific PIK3C3 kinase complexes .

What are the implications of PIK3C3 overexpression in transgenic models?

Transgenic animal models overexpressing PIK3C3 have provided crucial insights into its physiological functions and potential disease connections:

Liver Pathophysiology in PIK3C3 Transgenic Pigs:
Research with PIK3C3 transgenic pigs revealed significant hepatic alterations, including:

• Inflammatory infiltration and vacuolar formation in hepatocytes

• Increased apoptotic cells in liver tissue

• Upregulation of inflammatory markers (NF-κB, TGF-β1, TLR4, TNF-α, and IL-6)

• Enhanced LC3B and LAMP-1-positive cell populations, indicating increased autophagy

• Accelerated clearance of accumulated LC3II protein in fibroblast cells during starvation-induced autophagy

Molecular Mechanism Analysis:
The transgenic pig model helped elucidate that PIK3C3 overexpression promotes autophagy through mechanisms related to the activation of ULK1, AMBR1, DRAM1, and MTOR pathways. These molecular changes ultimately contributed to liver damage in the animals .

Experimental Design Considerations:
When studying PIK3C3 transgenic models, researchers should:

• Establish appropriate control groups (wild-type littermates)

• Perform comprehensive tissue analysis including histopathology and immunofluorescence

• Conduct cellular isolation for ex vivo studies (as done with porcine fibroblast cells)

• Validate gene expression changes via qPCR and protein levels via Western blotting

• Use starvation challenges (e.g., EBSS treatment) to assess autophagy dynamics

How can PIK3C3 kinase activity be distinguished from its role in autophagy regulation?

PIK3C3 possesses dual functions: lipid kinase activity (generating PtdIns3P) and protein kinase activity. Distinguishing between these functions requires specialized experimental approaches:

Dual Function Analysis:
Research has revealed that PIK3C3 contributes to Sertoli cell polarity and male fertility through both its autophagy regulatory role and its protein kinase activity. Specifically, PIK3C3 phosphorylates HDAC6 at site S59, marking it for degradation through the ubiquitin-proteasome pathway .

Experimental Strategies for Function Separation:

• Mutational Analysis: Generate PIK3C3 constructs with mutations in:

  • Lipid kinase domain (affecting PtdIns3P production)

  • Protein kinase active site (affecting protein phosphorylation)

• Pharmacological Discrimination:

  • VPS34-specific inhibitors (e.g., VPS34-IN1) that primarily affect lipid kinase activity

  • Broad-spectrum kinase inhibitors that affect both functions

• Substrate-Specific Assays:

  • PtdIns3P production assays (e.g., ELISA-based detection)

  • In vitro protein kinase assays with purified substrates

  • Phospho-specific antibodies for PIK3C3 targets (e.g., HDAC6-pS59)

Physiological Relevance:
The study of PIK3C3 in Sertoli cells demonstrated that through phosphorylation of HDAC6, PIK3C3 prevents the deacetylation of SCIN at site K189, thereby maintaining proper F-actin cytoskeleton organization. When PIK3C3 was deleted in the conditional knockout model, accumulated HDAC6 led to SCIN deacetylation and cytoskeletal disorganization .

How can microRNA regulation of PIK3C3 pathways be investigated?

MicroRNAs (miRNAs) provide an additional layer of regulation for PIK3C3 and its signaling pathways, offering potential biomarkers and therapeutic targets:

miRNA Interactions with PI3K Pathway:
Research has identified specific miRNAs that interact with PI3K pathway components:

• miR-105 directly interacts with proteins in the PI3K pathway, including IRS1 and PDK1

• miR-767 has been implicated in PI3K signaling

• Bimodal miRNAs may serve as biomarkers for patient stratification in cancer prognosis and drug response

Experimental Approaches:

• miRNA-Protein Interaction Verification:

  • Confirm downstream effects on protein targets (e.g., IRS1, PDK1, FOXO3)

  • Measure AGO2 binding to target mRNAs following miRNA treatment

  • Assess phosphorylation status of pathway components

• Cell Type Considerations:

  • Test multiple cell lines to determine whether miRNAs have universal mechanisms or cell-specific functions

  • A549 lung cancer cells and HepG2 liver cancer cells show different responses to miRNA treatment

• Functional Outcomes:

  • Measure cell survival and proliferation in response to miRNA modulation

  • Evaluate drug sensitivity changes when PI3K pathway miRNAs are manipulated

Combining FITC-conjugated PIK3C3 antibodies with miRNA analysis allows researchers to visualize how miRNA-mediated regulation affects PIK3C3 localization and complex formation in different cellular contexts.

What are common challenges when using PIK3C3 Antibody, FITC conjugated and how can they be addressed?

Researchers frequently encounter several technical challenges when working with FITC-conjugated PIK3C3 antibodies:

Signal Intensity Issues:

Specificity Concerns:

• Conduct parallel staining with multiple PIK3C3 antibodies recognizing different epitopes (e.g., N-terminal vs. C-terminal)

• Validate with recombinant PIK3C3 expression systems

• Perform siRNA knockdown to confirm signal reduction

Sample Preparation Optimization:

• For cell cultures: Fix with 4% paraformaldehyde rather than methanol to preserve FITC fluorescence

• For tissue sections: Use shorter fixation times (24-48 hours) and freshly cut sections

• Consider mild detergents (0.1% saponin) instead of Triton X-100 for gentler permeabilization

How can PIK3C3 Antibody, FITC conjugated be effectively used in multiplex immunofluorescence studies?

Multiplex immunofluorescence allows simultaneous visualization of PIK3C3 with other proteins, providing crucial insights into protein interactions and pathway dynamics:

Optimal Antibody Combinations:
When designing multiplex experiments with FITC-conjugated PIK3C3 antibodies, select secondary markers with spectrally distinct fluorophores:

• LC3B (autophagosome marker) with CY3 (red)

• LAMP-1 (lysosomal marker) with Alexa Fluor 647 (far red)

• ATG14 or UVRAG (PIK3C3 complex components) with Pacific Blue or similar

Sequential Staining Protocol:

• Block with 5% BSA/0.5% Triton X-100 for 30 minutes

• Apply FITC-conjugated PIK3C3 antibody (1:200-1:400) for 1 hour at room temperature or overnight at 4°C

• Wash extensively (3-5 times, 5 minutes each)

• Block again briefly (15 minutes)

• Apply unconjugated primary antibodies for other targets

• Wash thoroughly

• Apply spectrally compatible secondary antibodies

• Wash and mount with anti-fade medium containing DAPI

Confocal Analysis Approach:

• Use sequential scanning to minimize cross-channel bleed-through

• Acquire single-color controls for proper compensation

• Analyze colocalization using intensity correlation methods (Pearson's coefficient, Manders' overlap)

• Perform Z-stack imaging to assess 3D distribution and colocalization

The Liu et al. study successfully used this approach to demonstrate increased LC3B and LAMP-1 positive cells in liver tissue from PIK3C3 transgenic pigs compared to wild-type controls .

What are the optimal storage conditions for maintaining PIK3C3 Antibody, FITC conjugated activity?

Proper storage of FITC-conjugated PIK3C3 antibodies is crucial for maintaining long-term activity and fluorescence intensity:

Short-term Storage (up to 6 months):

• Store at 2-8°C (refrigerated)

• Protect from light by wrapping in aluminum foil or using opaque containers

• Do not freeze if stored short-term

Long-term Storage:

• Store at -20°C in small aliquots to prevent freeze-thaw cycles

• Add stabilizing proteins (1% BSA) if not already present in the formulation

• Use glycerol-containing buffers (up to 50%) to prevent freezing damage

Buffer Composition:
Optimal storage buffer typically contains:

• 0.01M TBS (pH 7.4)

• 1% BSA as stabilizer

• 0.03% Proclin300 or 0.09% sodium azide as preservative

• 50% Glycerol to prevent freeze-thaw damage

Critical Precautions:

• Minimize exposure to light at all times

• Avoid repeated freeze-thaw cycles by preparing small working aliquots

• Note that sodium azide is a POISONOUS AND HAZARDOUS SUBSTANCE requiring handling by trained staff

• Always briefly centrifuge vials after thawing to collect liquid at the bottom

Following these storage guidelines can extend antibody shelf-life to the standard expiry date (typically 6-12 months from production) .