PHACTR1 Antibody, FITC conjugated

What is PHACTR1 and why is it significant in scientific research?

PHACTR1 is a protein that contains four G-actin binding RPEL motifs and functions as a PP1-binding protein. It plays crucial roles in actin cytoskeleton reorganization, cell motility, and formation of actin stress fibers. PHACTR1 has gained significant research interest because:

• It is implicated in multiple human diseases including coronary artery disease, Parkinson's disease, cancer, and myocardial infarction

• Genetic variants in PHACTR1 are associated with arteriosclerotic cardiovascular disease

• A specific mutation (L519R) in PHACTR1 has been linked to multifocal epilepsy with infantile spasms

• PHACTR1 knockout can lead to instability of actin cytoskeleton reorganization, affecting processes like tumor cell migration

Understanding PHACTR1's regulatory mechanisms provides insights into fundamental cellular processes and potential therapeutic targets for various pathological conditions.

What are the key specifications of commercially available PHACTR1 Antibody, FITC conjugated?

Commercially available PHACTR1 Antibody, FITC conjugated products typically have the following specifications:

These specifications are derived from product information for commercially available PHACTR1 antibodies . When selecting an antibody for research, it's important to verify these specifications match your experimental requirements.

What are the recommended applications for PHACTR1 Antibody, FITC conjugated?

PHACTR1 Antibody, FITC conjugated is suitable for various research applications:

• Immunofluorescence microscopy: The FITC conjugation allows direct visualization of PHACTR1 in fixed cells and tissues without requiring secondary antibodies.

• Co-localization studies: Particularly useful for examining PHACTR1's relationship with F-actin when combined with phalloidin conjugated to a different fluorophore, as demonstrated in studies showing colocalization of PHACTR1 with F-actin .

• Flow cytometry: For quantitative analysis of PHACTR1 expression levels in cell populations.

• ELISA: For quantification of PHACTR1 protein levels in various samples .

• Double-staining experiments: FITC-conjugated PHACTR1 antibodies have been used effectively in double-staining protocols with F-actin markers to demonstrate their interaction .

The direct fluorophore conjugation simplifies experimental protocols, reduces background, and enables multi-color fluorescence experiments when studying PHACTR1's role in cytoskeletal dynamics and cellular processes.

What is the biological function of PHACTR1 that researchers are investigating?

Researchers are investigating several key biological functions of PHACTR1:

• Actin cytoskeleton regulation: PHACTR1 binds to actin monomers (G-actin) and plays a role in F-actin assembly and stress fiber formation. This function is critical for cell morphology and motility .

• Protein Phosphatase 1 (PP1) regulation: PHACTR1 interacts with and regulates PP1 activity. G-actin binding to PHACTR1 inhibits its interaction with PP1, creating a regulatory mechanism that links actin dynamics to phosphatase activity .

• Cellular migration and invasion: PHACTR1 promotes cell mobility through its effects on the actin cytoskeleton, as demonstrated in cancer studies where PHACTR1 silencing inhibited invasion and migration of cancer cells .

• Efferocytosis regulation: PHACTR1 facilitates efferocytosis (clearance of apoptotic cells) by decreasing PP1α-mediated myosin light chain (MLC) dephosphorylation, which is essential for maintaining cardiovascular health .

• Subcellular localization control: PHACTR1 shuttles between cytoplasm and nucleus in response to signals like serum stimulation, with its localization regulated by G-actin binding to RPEL motifs .

These functions highlight PHACTR1's importance in both normal cellular processes and pathological conditions, making it a valuable target for diverse research fields.

How should PHACTR1 Antibody, FITC conjugated be stored and handled?

Proper storage and handling of PHACTR1 Antibody, FITC conjugated is crucial for maintaining its functionality:

• Storage temperature: Upon receipt, store at -20°C or -80°C as specified by the manufacturer .

• Avoid freeze-thaw cycles: Repeated freezing and thawing significantly reduces antibody activity. Upon first thawing, prepare working aliquots to minimize future freeze-thaw cycles.

• Buffer conditions: The antibody is typically supplied in a buffer containing preservative (0.03% Proclin 300), stabilizers (50% Glycerol), and buffering agents (0.01M PBS, pH 7.4) .

• Light protection: FITC is sensitive to photobleaching, so protect the antibody from prolonged exposure to light during handling and storage.

• Working dilution preparation: Prepare working dilutions just before use and discard any unused diluted antibody.

• Temperature for handling: When using the antibody, keep it on ice or at 4°C.

• Contamination prevention: Use sterile pipette tips and tubes when handling to prevent contamination.

• Quality control: Check for signs of degradation (precipitation, color change) before use.

Following these guidelines will help ensure optimal antibody performance and reproducible experimental results.

How can PHACTR1 Antibody, FITC conjugated be utilized to study the role of PHACTR1 in actin cytoskeleton dynamics?

PHACTR1 Antibody, FITC conjugated provides a powerful tool for investigating actin cytoskeleton dynamics through several sophisticated approaches:

• Co-localization analysis with F-actin markers:

  • Double stain cells with PHACTR1-FITC antibody and F-actin markers like phalloidin-TRITC

  • Analyze spatial correlation between PHACTR1 and F-actin structures using confocal microscopy

  • Quantify colocalization using Pearson's or Mander's coefficients

  • Research has confirmed that PHACTR1 colocalizes with F-actin in multiple cell types

• Actin reorganization studies:

  • Manipulate PHACTR1 expression (knockdown/overexpression) and visualize resulting changes in F-actin organization

  • Studies show PHACTR1 silencing sharply reduces F-actin formation, while overexpression increases F-actin structures

  • Quantify F-actin intensity, stress fiber thickness, and morphological changes

• Dynamic processes visualization:

  • Study PHACTR1 localization during actin-dependent processes like cell migration, division, or phagocytosis

  • PHACTR1 silencing in macrophages causes defects in actin cup formation around engaged apoptotic cells

• Mutation impact assessment:

  • Compare PHACTR1 localization and actin organization between wild-type and mutant PHACTR1 (e.g., RPEL motif mutations)

  • The L519R mutation in PHACTR1 exhibits altered subcellular localization and increased ability to induce cytoskeletal rearrangements

• G-actin/F-actin ratio correlation:

  • Combine PHACTR1-FITC staining with techniques that distinguish G-actin vs. F-actin pools

  • Correlate PHACTR1 localization with cellular G-actin/F-actin ratios under various conditions

• Pharmacological intervention studies:

  • Use actin-disrupting agents like Swinholide A as controls

  • Research shows Swinholide A can block F-actin formation enhanced by PHACTR1 overexpression

  • Monitor PHACTR1 redistribution after cytoskeletal disruption

These approaches enable detailed investigation of PHACTR1's mechanistic role in regulating actin dynamics, which is crucial for understanding its functions in cell mobility, morphology, and pathological conditions.

What experimental methods can demonstrate the relationship between PHACTR1's G-actin binding capacity and its function?

The relationship between PHACTR1's G-actin binding capacity and its function can be demonstrated through several sophisticated experimental approaches:

• In vitro G-actin binding assays:

  • Use purified PHACTR1 (wild-type and mutants) to measure G-actin binding affinity

  • Research shows mutations like L519R reduce RPEL3 motif's G-actin binding affinity approximately 3-fold

  • Compare binding coefficients between wild-type and mutated PHACTR1 proteins

• Competitive binding experiments:

  • Assess competition between G-actin and PP1 for binding to PHACTR1

  • Studies demonstrate G-actin binding to PHACTR1 inhibits its interaction with PP1, whose binding site overlaps with RPEL3 motif

  • Use co-immunoprecipitation to demonstrate mutants with reduced G-actin binding show increased PP1 binding

• Subcellular localization studies:

  • Compare localization patterns of wild-type vs. G-actin binding-deficient mutants

  • Wild-type PHACTR1 is predominantly cytoplasmic in resting cells, while mutants with reduced G-actin binding exhibit increased nuclear localization

  • Use FITC-conjugated antibodies to visualize this differential localization

• Functional outcome assessments:

  • Examine downstream effects of altered G-actin binding

  • Mutants that can't bind G-actin efficiently (e.g., R507A) induce aberrant F-actin structures

  • Quantify phenotypic differences in actin organization, cell morphology, and behavior

• Signal-responsive dynamics:

  • Monitor PHACTR1 localization during serum stimulation or Rho-activation

  • Serum stimulation induces PHACTR1 nuclear accumulation through Rho-actin signaling

  • Compare wild-type vs. G-actin binding mutants in their response to these signals

• MLC phosphorylation analysis:

  • Measure phosphorylated MLC levels as a downstream marker of PHACTR1 activity

  • Binding of apoptotic cells to macrophages induces MLC phosphorylation in a PHACTR1-dependent manner

  • Compare pMLC levels in cells expressing wild-type vs. G-actin binding-deficient PHACTR1

These methodologies collectively demonstrate how PHACTR1's G-actin binding capacity regulates its subcellular localization, interaction with PP1, and subsequent effects on actin cytoskeleton organization and cellular functions.

How does PHACTR1 expression influence cellular processes in different disease models?

PHACTR1 expression significantly impacts cellular processes across various disease models, with distinct mechanistic patterns emerging:

• Cardiovascular disease models:

  • Genetic deficiency of Phactr1 promotes atherosclerosis in mouse models

  • PHACTR1-deficient macrophages show impaired efferocytosis (clearance of apoptotic cells)

  • The CAD-risk allele (rs9349379-GG) correlates with lower PHACTR1 expression in human monocyte-derived macrophages

  • Mechanistically, PHACTR1 facilitates efferocytosis by maintaining phosphorylated myosin light chain (MLC), which enables proper actin cytoskeleton remodeling

• Cancer progression models:

  • PHACTR1 expression is elevated in metastatic or larger papillary thyroid carcinoma (PTC) tissues

  • More mobile cancer cell lines (K1 cells) show higher PHACTR1 expression compared to less mobile lines (TPC-1)

  • PHACTR1 silencing inhibits invasion, migration, and tumorigenicity in cancer cells

  • Mechanistically, PHACTR1 promotes F-actin assembly, which drives cancer cell mobility

  • Treatment with actin-disrupting agent Swinholide A blocks the enhanced invasion and migration caused by PHACTR1 overexpression

• Neurological disorder models:

  • A heterozygous L519R mutation in PHACTR1 is associated with multifocal epilepsy with infantile spasms

  • This mutation reduces G-actin binding affinity and increases PHACTR1's propensity to form complexes with PP1

  • The mutant exhibits altered subcellular localization (increased nuclear presence) and enhanced ability to induce cytoskeletal rearrangements

  • These properties suggest the mutation is activating in nature, potentially affecting neuronal excitability

• Functional impact on cell behavior:

  • PHACTR1 knockout in breast cancer cells weakens TGF-β-induced tumor cell migration

  • PHACTR1 knockout can lead to defective neuronal migration during corticogenesis

  • In macrophages, PHACTR1 is required for proper actin cup formation during efferocytosis

These findings demonstrate PHACTR1's context-dependent roles in disease processes, primarily through its regulation of the actin cytoskeleton and subsequent effects on cell migration, phagocytosis, and tissue remodeling.

What methodological approaches can resolve contradictory findings about PHACTR1 function in different experimental systems?

Researchers face seemingly contradictory findings about PHACTR1 function across different experimental systems. The following methodological approaches can help resolve these contradictions:

• Cell-type specific analysis:

  • Different cell types may exhibit opposing PHACTR1 functions

  • Use the same PHACTR1 antibody across multiple cell types

  • For example, PHACTR1 appears protective in macrophages against atherosclerosis but promotes mobility in cancer cells

  • Isolate primary cells from multiple tissues to compare PHACTR1 function systematically

• Context-dependent signaling pathway mapping:

  • Identify different downstream pathways activated by PHACTR1 in various contexts

  • In macrophages, PHACTR1 activates CREB signaling via directly binding to CREB, upregulating CREB phosphorylation and inducing KLF4 expression

  • In fibroblasts and cancer cells, PHACTR1 regulates actin stress fiber formation

  • Use phospho-proteomics to create comprehensive signaling maps

• Integrating genetic variant data with functional outcomes:

  • Different genetic variants of PHACTR1 may have opposite effects

  • Compare functional outcomes between different PHACTR1 variants

  • The L519R mutation appears activating, increasing PP1 binding and cytoskeletal rearrangements

  • The CAD-risk allele (rs9349379-GG) results in lower PHACTR1 expression and impaired function

• Distinguishing direct vs. indirect effects:

  • Use rapid induction systems (optogenetics, chemical dimerization) to isolate immediate PHACTR1 effects

  • Separate effects of nuclear vs. cytoplasmic PHACTR1 using localization-restricted variants

  • Apply temporal analysis to distinguish primary from secondary effects

• Validation across multiple experimental models:

  • Compare in vitro results with in vivo models

  • Use both gain-of-function and loss-of-function approaches

  • Example: PHACTR1 silencing reduces xenograft size in vivo, confirming in vitro observations about its role in cancer progression

• Standardized quantification methods:

  • Develop consistent metrics for measuring PHACTR1-dependent processes

  • For F-actin analysis: standardize staining protocols, image acquisition parameters, and quantification algorithms

  • For migration: adopt uniform methods (wound healing, transwell assays) with consistent quantification

• Integration of multi-omics data:

  • Combine transcriptomics, proteomics, and functional data

  • Genome-wide transcriptional profiling has revealed PHACTR1 as a regulatory target in some conditions

  • Create comprehensive models that account for context-specific factors

These approaches help create a more nuanced understanding of PHACTR1's context-dependent functions and resolve apparent contradictions in the literature.

What controls and validation steps are critical when using PHACTR1 Antibody, FITC conjugated for immunofluorescence studies?

When using PHACTR1 Antibody, FITC conjugated for immunofluorescence studies, implementing rigorous controls and validation steps is essential for generating reliable and reproducible results:

• Antibody specificity validation:

  • Genetic knockdown/knockout control: Compare staining in PHACTR1 knockdown/knockout cells to wild-type cells

  • Overexpression control: Verify increased signal in cells overexpressing PHACTR1

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide to demonstrate specific blocking

  • Isotype control: Use FITC-conjugated isotype-matched non-specific IgG at equivalent concentration

• Technical controls for immunofluorescence:

  • Secondary antibody-only control: Even with direct conjugates, this helps assess autofluorescence

  • Single-color controls: For spectral overlap correction when performing multi-color experiments

  • Fixation method comparison: Test multiple fixation methods as PHACTR1 epitope accessibility may vary

  • Permeabilization optimization: Systematically test permeabilization conditions to balance antigen accessibility and structural preservation

• Biological validation approaches:

  • Correlation with expected localization patterns: PHACTR1 shows cytoplasmic localization in resting cells and nuclear accumulation after serum stimulation

  • Co-localization with known interaction partners: Verify co-localization with F-actin structures using phalloidin

  • Response to stimuli: Confirm PHACTR1 relocalization following serum stimulation

  • Mutant comparisons: Use cells expressing PHACTR1 with mutations affecting localization (e.g., RPEL motif mutations)

• Quantification validation:

  • Replicate analysis: Perform analysis across multiple fields, samples, and experimental days

  • Blinded quantification: Have images quantified by researchers blinded to experimental conditions

  • Multiple quantification methods: Apply different measurement approaches (intensity, localization ratio, co-localization coefficients)

  • Statistical validation: Ensure appropriate statistical tests and sample sizes

• Technical optimization for FITC detection:

  • Photobleaching controls: Include time-series controls to account for FITC photobleaching

  • Autofluorescence correction: Use unstained samples to establish baseline autofluorescence

  • Optical setup optimization: Adjust filter sets and exposure settings for optimal FITC detection

  • Mounting media selection: Use anti-fade mounting media specifically compatible with FITC

• Protocol standardization documentation:

  • Document all parameters: antibody dilution (typically 1:40 to 1:500), incubation times, washing steps

  • Record imaging parameters: exposure time, gain settings, objective specifications

  • Maintain consistent protocols across experimental comparisons

These comprehensive controls and validation steps ensure that findings related to PHACTR1 localization, expression, and function are reliable and biologically meaningful.

How can researchers effectively use PHACTR1 Antibody, FITC conjugated to study its role in efferocytosis?

Studying PHACTR1's role in efferocytosis (the clearance of apoptotic cells) requires specialized experimental approaches that leverage FITC-conjugated PHACTR1 antibodies:

• Efferocytosis assay establishment:

  • Generate labeled apoptotic cells (ACs) using PKH26 dye

  • Prepare macrophages (human monocyte-derived or bone marrow-derived)

  • Measure efferocytosis by quantifying internalized ACs

  • Distinguish binding from internalization using temperature-controlled conditions

• PHACTR1 expression manipulation:

  • Use siRNA silencing or CRISPR-Cas9 knockout of PHACTR1 in macrophages

  • Research shows PHACTR1 silencing leads to impaired efferocytosis under both basal and inflammatory conditions

  • Create rescue systems by re-expressing wild-type or mutant PHACTR1

• Visualizing PHACTR1 dynamics during efferocytosis:

  • Use PHACTR1-FITC antibody to track protein localization during AC engagement

  • Combine with F-actin markers to visualize phagocytic cup formation

  • PHACTR1 silencing in macrophages causes defects in the formation of typical wide-mouth actin cups around engaged ACs

• Mechanistic pathway investigation:

  • Monitor myosin light chain (MLC) phosphorylation during efferocytosis

  • AC binding to macrophages induces MLC phosphorylation in a PHACTR1-dependent manner

  • Track PP1α activity in relation to PHACTR1 localization and function

• Genotype-phenotype correlation studies:

  • Compare efferocytosis capacity between macrophages from individuals with different PHACTR1 genotypes

  • HMDMs carrying CAD-risk allele (GG) show lower PHACTR1 levels and impaired efferocytosis compared to AA or AG genotypes

• Multi-parameter analysis:

  • Combine PHACTR1-FITC staining with markers for:

    • Phosphorylated MLC to track activation status

    • PP1α to monitor phosphatase recruitment

    • Actin regulatory proteins (Arp2/3, cofilin) to assess cytoskeletal dynamics

  • Perform time-course analysis capturing all stages of efferocytosis

• Inflammation context evaluation:

  • Test efferocytosis under both basal and inflammatory conditions (IFN-γ/LPS treatment)

  • Research shows PHACTR1 is required for efficient efferocytosis under both conditions

These methodological approaches enable detailed investigation of PHACTR1's role in efferocytosis, a process crucial for tissue homeostasis whose impairment drives atherosclerosis progression and complications.

What are the considerations for using PHACTR1 Antibody, FITC conjugated in multi-color immunofluorescence experiments?

Multi-color immunofluorescence experiments involving PHACTR1 Antibody, FITC conjugated require careful consideration of multiple technical and experimental factors:

Implementing these considerations ensures reliable and informative multi-color immunofluorescence experiments that effectively reveal PHACTR1's interactions and functions in cellular contexts.

How can researchers assess the impact of PHACTR1 mutations on protein function using FITC-conjugated antibodies?

Assessing the impact of PHACTR1 mutations on protein function using FITC-conjugated antibodies requires sophisticated experimental designs that integrate multiple methodological approaches:

• Mutation-specific expression systems:

  • Generate cell lines expressing wild-type vs. mutant PHACTR1 variants

  • Document mutations of interest:

    • L519R: associated with multifocal epilepsy

    • R507A: affects the core RPEL3 arginine, severely reducing G-actin binding

    • RPEL motif mutations: affect G-actin binding and subcellular localization

• Subcellular localization analysis:

  • Use PHACTR1-FITC antibody to compare localization patterns

  • Wild-type PHACTR1 is predominantly cytoplasmic in resting cells

  • PHACTR1 L519R and R507A mutants exhibit increased nuclear localization

  • Quantify nuclear/cytoplasmic distribution ratios across multiple cells

• G-actin binding assessment:

  • The L519R mutation reduces RPEL3 affinity for G-actin approximately 3-fold

  • R507A mutation reduces affinity 18-fold

  • Correlate binding affinity changes with subcellular localization patterns

• PP1 interaction studies:

  • PHACTR1 L519R exhibits greatly increased interaction with PP1

  • Perform co-immunoprecipitation experiments comparing wild-type and mutant variants

  • Visualize PHACTR1-PP1 co-localization using double immunofluorescence

• Cytoskeletal impact evaluation:

  • Assess F-actin organization in cells expressing different PHACTR1 variants

  • PHACTR1 L519R and R507A induce thick F-actin fibers and F-actin foci even in resting cells

  • Quantify F-actin structures (density, thickness, organization) using appropriate imaging software

• Stimulus response characterization:

  • Compare how wild-type vs. mutant PHACTR1 respond to serum stimulation

  • Wild-type PHACTR1 weakly induces F-actin rearrangements only upon serum stimulation

  • Mutants show constitutive activity or enhanced response to stimulation

• Functional rescue experiments:

  • Silence endogenous PHACTR1 and re-express wild-type or mutant variants

  • Assess restoration of normal function (e.g., actin organization, cell migration)

  • Determine which domains are critical for specific functions

• Domain-specific mutant analysis:

  • Create and analyze mutations in different functional domains:

    • RPEL motifs: affect G-actin binding

    • PP1-binding domain: affect phosphatase regulation

    • NLS regions: affect nuclear localization

• Correlation with pathological outcomes:

  • Link functional changes to disease mechanisms

  • The L519R mutation's increased PP1 binding may affect neuronal excitability in epilepsy

  • Document how specific mutations affect cell-type specific functions

These approaches allow researchers to comprehensively characterize how mutations alter PHACTR1's interactions, localization, and functional outcomes, providing insights into both normal function and disease mechanisms.

What techniques can be combined with PHACTR1 immunofluorescence to study its role in cytoskeletal dynamics?

To comprehensively study PHACTR1's role in cytoskeletal dynamics, researchers can combine PHACTR1 immunofluorescence with multiple complementary techniques:

• Advanced microscopy methods:

  • Live cell imaging: Track real-time changes in cytoskeleton using LifeAct-RFP combined with fixed timepoint PHACTR1-FITC staining

  • Super-resolution microscopy (STORM, PALM, SIM): Resolve fine cytoskeletal structures beyond diffraction limit

  • FRAP (Fluorescence Recovery After Photobleaching): Measure actin turnover rates in PHACTR1-manipulated cells

  • TIRF microscopy: Visualize PHACTR1 and actin interactions at the cell surface

• Biochemical assays:

  • Co-immunoprecipitation: Verify PHACTR1's interaction with actin and PP1

  • F/G-actin fractionation: Quantify F/G-actin ratios in cells with altered PHACTR1 expression

  • In vitro actin polymerization assays: Assess how PHACTR1 affects actin assembly kinetics

  • Phosphorylation analysis: Monitor MLC phosphorylation, which PHACTR1 maintains by regulating PP1

• Cytoskeletal perturbation approaches:

  • Actin-disrupting agents: Use Swinholide A to block PHACTR1-induced F-actin formation

  • PP1 inhibitors: Determine which PHACTR1 effects require PP1 activity

  • Myosin inhibitors: Test dependency on actomyosin contractility

• Molecular manipulation strategies:

  • Domain-specific mutations: Create PHACTR1 variants with altered G-actin or PP1 binding

  • Inducible expression systems: Control PHACTR1 levels with temporal precision

  • Subcellular targeting: Force PHACTR1 localization to specific compartments

• Dynamic process analysis:

  • Migration assays: Wound healing, single-cell tracking, chemotaxis chambers

  • Invasion assays: Transwell chambers with Matrigel coating

  • Efferocytosis assays: Quantify phagocytic cup formation and AC internalization

• Quantitative image analysis:

  • F-actin morphometry: Measure stress fiber thickness, density, orientation

  • Cell shape parameters: Quantify changes in cell spreading, elongation, polarization

  • Protrusion dynamics: Analyze lamellipodia/filopodia formation and lifetime

  • Colocalization metrics: Pearson's correlation coefficient between PHACTR1 and F-actin

• Mechanistic pathway investigation:

  • Rho GTPase activity assays: Determine relationship with RhoA signaling

  • Phosphoproteomic analysis: Identify cytoskeletal substrates affected by PHACTR1-PP1

  • Interaction screens: Identify additional PHACTR1 binding partners

These complementary approaches create a comprehensive understanding of PHACTR1's multifaceted roles in cytoskeletal regulation, from molecular interactions to cellular behaviors, providing mechanistic insights applicable to both normal function and disease states.

How can researchers quantitatively analyze PHACTR1's effect on F-actin formation using immunofluorescence data?

Quantitative analysis of PHACTR1's effect on F-actin formation using immunofluorescence data requires rigorous methodological approaches and appropriate analytical tools: