PACSIN2 Antibody

What is PACSIN2 and why is it important in cellular research?

PACSIN2 belongs to the BAR (Bin/Amphiphysin/Rvs) domain family of proteins involved in membrane remodeling and cytoskeletal interactions. It's ubiquitously expressed (unlike other PACSIN family members) and plays critical roles in membrane curvature, endocytosis, and actin cytoskeleton regulation. Research shows PACSIN2 is particularly important in viral pathogenesis, notably in HIV-1 cell-to-cell transmission through its interaction with the actin cytoskeleton via its SH3 domain that binds regulators like WASP and N-WASP . When designing experiments to study membrane dynamics or viral trafficking, targeting PACSIN2 provides insights into fundamental cellular processes that influence disease progression.

What are the most validated applications for PACSIN2 antibodies?

Based on published research and validation data, PACSIN2 antibodies have been extensively validated for:

For initial characterization of PACSIN2 in a new experimental system, begin with western blotting to confirm protein expression and molecular weight (typically observed at 60-65 kDa) , followed by localization studies using immunofluorescence.

Which cell and tissue types show reliable PACSIN2 detection?

PACSIN2 antibodies have demonstrated reliable detection in:

Cell lines:

• HEK-293 cells

• NIH/3T3 cells

• HepG2 cells

• MOLT3 CD4+ cells (in HIV research)

Tissues:

• Mouse heart tissue

• Mouse lung tissue

• Human breast cancer tissue

• Human skin tissue

When working with new cell types or tissues, optimize antigen retrieval conditions; for example, PACSIN2 detection in human tissues works best with TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative .

How does PACSIN2 function differ from other PACSIN family members in experimental settings?

Unlike PACSIN1 (predominantly neuronal) and PACSIN3 (muscle-enriched), PACSIN2 is ubiquitously expressed, making it relevant across diverse experimental systems. PACSIN2's F-BAR domain exhibits membrane binding and curvature-inducing properties, demonstrated by its ability to cause in vitro tubulation of liposomes .

When designing experiments comparing PACSIN family members, consider:

• PACSIN2 functions in non-neuronal contexts where membrane remodeling is critical

• PACSIN2's unique interactions with viral proteins (like HIV-1 Gag) that aren't observed with other family members

• PACSIN2's role in connecting membrane dynamics to actin cytoskeleton through its SH3 domain interactions

For accurate functional comparison experiments, ensure antibody specificity to prevent cross-reactivity with other PACSIN family members, particularly in neural tissues where multiple isoforms may be expressed.

What are the challenges in studying PACSIN2-dependent processes in viral pathogenesis?

Investigating PACSIN2's role in viral pathogenesis, particularly HIV-1 spreading, presents several methodological challenges:

• Distinguishing direct vs. indirect effects: PACSIN2 depletion severely impairs HIV-1 spreading in T cell lines and primary PBMCs, but PACSIN2 is dispensable for single-cycle replication with cell-free virus . This suggests PACSIN2 specifically mediates cell-to-cell transmission rather than viral budding.

• Temporal dynamics: Virus spreading experiments require extended timeframes (9+ days) to observe PACSIN2 depletion effects, necessitating stable knockdown systems rather than transient approaches .

• Domain-specific functions: Rescue experiments revealed that PACSIN2's SH3 domain interaction with actin regulators is critical for HIV-1 spreading . When designing constructs for rescue experiments, domain-specific mutations rather than complete protein depletion provide more mechanistic insights.

To overcome these challenges, implement combined approaches using:

• Stable shRNA knockdown with multiple targeting sequences to confirm specificity

• Rescue experiments with domain-specific mutants

• Temporally extended experimental designs (monitoring viral spread over 5-9 days)

• Complementary cell-free and cell-to-cell transmission assays

How do post-translational modifications affect PACSIN2 antibody detection and function?

PACSIN2 undergoes several post-translational modifications that can impact antibody recognition and protein function:

• Ubiquitination: PACSIN2 incorporation into HIV-1 virus-like particles correlates with Gag-ubiquitin conjugates, suggesting PACSIN2 binds ubiquitin . This modification may mask epitopes recognized by certain antibodies.

• Phosphorylation: Regulatory phosphorylation can alter PACSIN2 conformation and function. When using phospho-specific antibodies, consider:

  • Preserving phosphorylation status using phosphatase inhibitors in lysate preparation

  • Using appropriate controls (phosphatase-treated samples) to validate specificity

• Molecular weight shifts: The observed molecular weight of PACSIN2 (60-65 kDa) differs from the calculated weight (56 kDa) , likely due to these modifications.

For comprehensive PACSIN2 analysis, consider using multiple antibodies targeting different epitopes, particularly when studying conditions that might alter post-translational modification patterns.

What are the optimal conditions for PACSIN2 detection by immunofluorescence?

For successful PACSIN2 immunofluorescence staining:

• Fixation protocol:

  • 4% paraformaldehyde (10-15 minutes at room temperature) preserves PACSIN2 localization at membrane structures

  • Avoid methanol fixation which can disrupt membrane-associated epitopes

• Antibody dilution and incubation:

  • Use 1:200-1:800 dilution range, optimizing for your specific cell type

  • Incubate primary antibody overnight at 4°C to maximize specific binding

  • For NIH/3T3 cells (validated system), begin with 1:400 dilution

• Visualization considerations:

  • PACSIN2 typically shows peripheral/membrane localization pattern in many cell types

  • Co-staining with membrane markers or actin cytoskeleton components provides contextual information

  • Use confocal microscopy for optimal resolution of membrane-associated structures

• Controls:

  • Include PACSIN2-depleted cells as negative controls

  • Consider co-staining with markers of specific membrane compartments to establish localization pattern

How can I design effective PACSIN2 knockdown/knockout experiments?

Based on published research with PACSIN2 depletion:

• shRNA approach:

  • Multiple independent shRNAs targeting different PACSIN2 regions have successfully depleted expression in MOLT3 and MOLT4 cl.8 cells

  • Stable knockdown is preferable for long-term experiments like viral spreading assays

  • Validate knockdown efficiency by Western blot before phenotypic analysis

• Rescue experiment design:

  • Use shRNA-resistant PACSIN2 constructs (containing silent mutations in the shRNA target sequence)

  • Tagged versions (HA-PACSIN2) allow distinction from endogenous protein

  • Domain-specific mutants (particularly SH3 domain) provide mechanistic insights

• Functional validation:

  • For HIV-1 studies, virus spreading assays over 5-9 days provide clear phenotypic readout

  • In primary cells (PBMCs), ensure efficient transduction methods for PACSIN2 depletion

  • Monitor both virus production and cell-to-cell transmission separately

• Phenotypic analysis timeframe:

  • Allow sufficient time for phenotype development (9+ days for HIV spreading)

  • Include time-course analysis rather than single endpoints

What troubleshooting strategies address common issues with PACSIN2 antibody applications?

Western Blotting Issues:

Immunohistochemistry Challenges:

Immunoprecipitation Optimization:

For successful PACSIN2 immunoprecipitation, use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate . Crosslinking techniques may be beneficial when studying PACSIN2's transient interactions with actin cytoskeleton components or viral proteins.

How does PACSIN2 contribute to HIV-1 pathogenesis in current research models?

PACSIN2 plays a specialized role in HIV-1 pathogenesis through several mechanisms:

• Viral spreading: PACSIN2 is critical for cell-to-cell transmission of HIV-1, the predominant mode of viral spread between T cells. Depletion of PACSIN2 severely impairs HIV-1 spreading in both T cell lines and primary human PBMCs .

• Recruitment mechanism: HIV-1 p6 domain specifically recruits PACSIN2 into virus-like particles. This recruitment occurs independently of ESCRT factors TSG101 or ALIX, suggesting a parallel pathway .

• Cytoskeletal connection: PACSIN2's SH3 domain mediates interactions with actin polymerization regulators WASP and N-WASP. Mutating this domain prevents restoration of HIV-1 spreading in PACSIN2-depleted cells, demonstrating the essential nature of this connection .

• Methodological approach: When studying PACSIN2 in HIV pathogenesis, distinguish between:

  • Cell-free virus production (relatively unaffected by PACSIN2)

  • Cell-to-cell transmission (critically dependent on PACSIN2)

This research demonstrates how specialized cellular membrane-cytoskeleton connectors can be exploited by viruses for efficient spreading, providing potential therapeutic targets.

What experimental systems best demonstrate PACSIN2 membrane remodeling activities?

To study PACSIN2's membrane remodeling functions:

• In vitro liposome tubulation assays: PACSIN2's F-BAR domain induces membrane curvature, observable as tubulation of liposomes in reconstituted systems .

• Cellular models:

  • NIH/3T3 cells show reliable PACSIN2 localization at the cell periphery by immunofluorescence

  • T24 cells demonstrate clear PACSIN2 peripheral localization

• Visualization approaches:

  • Super-resolution microscopy to visualize membrane curvature

  • Live-cell imaging with fluorescently tagged PACSIN2 to observe dynamic membrane remodeling

  • Correlative light-electron microscopy to connect PACSIN2 localization with membrane ultrastructure

• Functional assays:

  • Endocytosis tracking of specific cargoes (e.g., N-cadherin internalization)

  • Membrane dynamics during cell spreading or migration

  • Viral particle assembly and budding systems

When designing these experiments, consider PACSIN2's interactions with both membrane components and cytoskeletal elements that may influence its membrane remodeling capacity.

How can researchers distinguish between the roles of PACSIN2 and other BAR domain proteins?

BAR domain proteins share structural similarities but exhibit functional specialization. To differentiate PACSIN2's specific functions:

• Domain-specific approach:

  • PACSIN2 contains an F-BAR domain (membrane binding/curvature) and an SH3 domain (protein-protein interactions)

  • Create chimeric constructs swapping domains between PACSIN2 and other BAR proteins to identify domain-specific functions

• Interaction network analysis:

  • PACSIN2 specifically interacts with WASP/N-WASP through its SH3 domain

  • Compare immunoprecipitation profiles of different BAR proteins to identify unique binding partners

• Cellular localization patterns:

  • Use co-localization studies with multiple BAR proteins to identify compartment-specific enrichment

  • PACSIN2 typically shows peripheral/membrane localization in cells like T24

• Sequential depletion studies:

  • Knockdown individual BAR proteins and assess compensation by other family members

  • Double knockdown experiments to identify redundant versus unique functions

• Temporal dynamics:

  • Live-cell imaging with different fluorescently-tagged BAR proteins may reveal distinct recruitment kinetics during processes like endocytosis or membrane remodeling

By systematically comparing PACSIN2 with other BAR proteins like Angiomotin (which functions during early HIV-1 budding) , researchers can establish the unique spatiotemporal contributions of each family member to membrane dynamics.