xirp1 Antibody

What is XIRP1 and what cellular functions does it regulate?

XIRP1 is a multi-domain cytoskeletal protein that localizes to fibroblast focal adhesions and macrophage podosomes . It contains actin-binding xin repeat domains and proline-rich domains that interact with key cytoskeletal components including VASP, F-actin, and Filamin C . XIRP1 expression is induced by immune cytokines, particularly interferon-gamma (IFN-γ), and during bacterial infections with pathogens such as Listeria, Shigella, and Salmonella .

Functionally, XIRP1 plays important roles in:

• Structural organization of podosomes in macrophages

• Cell-autonomous immunity mechanisms

• Cardiac conduction and heart development

• Potential tumor suppression in glioblastoma

Human XIRP1 exists in three distinct isoforms with varying domain structures, which influences its cellular functions and protein interactions .

Which antibody applications are most effective for XIRP1 detection in different cell types?

For macrophage podosome studies, immunofluorescence using rabbit polyclonal antibodies shows excellent results . Most commercially available antibodies target the C-terminal region (amino acids 1338-1367) of human XIRP1 , though antibodies against different epitopes are also available for specialized applications. When selecting an antibody, consider the specific XIRP1 isoform and subcellular compartment relevant to your research question.

How does XIRP1 localization differ between fibroblasts and macrophages?

XIRP1 displays distinct localization patterns that reflect its cell type-specific functions:

In fibroblasts:

• Primarily localizes to focal adhesions (cell-matrix adhesion complexes)

• Expression requires both IFN-γ stimulation and additional signals like bacterial infection or IL-1β

• Forms discrete puncta at the cell-substrate interface

In macrophages:

• Concentrates in podosomes, forming specialized actin-rich adhesion structures

• Super-resolution structured illumination microscopy (SIM) reveals conical XIRP1 structures distributed at the base of macrophages

• Forms part of the podosome network facilitating macrophage adhesion and migration

• Expression can be induced by IFN-γ alone in PMA-differentiated THP-1 macrophages

During bacterial infection with Listeria monocytogenes, XIRP1 exhibits additional localization patterns:

• Recruited to bacterial surfaces in an ActA-dependent manner

• Forms an encircling pattern around replicating bacteria

• Associates with actin tails during bacterial motility

This differential localization must be considered when designing immunostaining protocols and interpreting antibody staining patterns.

What are the optimal fixation protocols for preserving XIRP1 epitopes in immunofluorescence studies?

For optimal XIRP1 epitope preservation in immunofluorescence studies, researchers should implement the following protocol based on successful detection methods in published studies :

• Fixation:

  • Use 4% paraformaldehyde in PBS for 15-20 minutes at room temperature

  • Avoid methanol fixation which can disrupt cytoskeletal epitopes

• Permeabilization:

  • Use 0.1-0.2% Triton X-100 in PBS for 5-10 minutes

  • For super-resolution microscopy, shorter permeabilization times (3-5 minutes) may reduce background

• Blocking:

  • Block with 3-5% BSA or normal serum in PBS for at least 30 minutes

  • Include 0.1% saponin in blocking buffer for macrophage samples to enhance antibody penetration

• Special considerations for infection studies:

  • Fix infected cells at specific time points post-infection to capture dynamic recruitment events

  • Use gentler permeabilization for bacteria-infected samples to preserve bacterial morphology

  • Consider paraformaldehyde-glutaraldehyde mixture (4% PFA, 0.1% glutaraldehyde) for preserving actin structures during bacterial motility

• Antibody incubation:

  • Use primary antibodies at 1:50-1:200 dilution in blocking buffer

  • Incubate overnight at 4°C for optimal signal-to-noise ratio

  • For multi-color staining, apply antibodies sequentially if raised in the same host species

These protocols have successfully visualized XIRP1 in both standard confocal and super-resolution microscopy applications .

How do different XIRP1 isoforms impact antibody selection for experimental design?

Human XIRP1 exists in three isoforms with varying domain structures, significantly impacting antibody selection strategy . The diagram of human XIRP1 isoforms shows different arrangements of actin-binding xin repeat domains, proline-rich domains, and interaction regions for VASP, F-actin, and Filamin C .

When selecting antibodies:

• Consider epitope location relative to isoform structure:

  • C-terminal antibodies (aa 1338-1367) detect all full-length isoforms

  • N-terminal antibodies may miss truncated isoforms

  • Middle region antibodies have isoform-specific reactivity patterns

• Match antibody to experimental question:

  • For total XIRP1 detection: use antibodies against conserved regions

  • For isoform discrimination: select antibodies against unique regions

  • For functional studies: choose antibodies that don't interfere with critical interaction domains

• Validate antibody specificity:

  • Use CRISPR-Cas9 XIRP1 knockout cells as negative controls

  • Test multiple antibodies targeting different epitopes

  • Confirm expected molecular weight detection for each isoform

• Consider species cross-reactivity:

  • Human-specific antibodies for clinical samples

  • Cross-reactive antibodies for animal models

  • Verify epitope conservation across species for comparative studies

Successful studies have employed both N-terminal (aa 1-532) and C-terminal antibodies, with validation through CRISPR-Cas9 XIRP1 deletions to confirm specificity .

What are the current limitations in detecting XIRP1 interactions with cytoskeletal components during bacterial infection?

Studying XIRP1 interactions with cytoskeletal components during bacterial infection presents several methodological challenges:

• Dynamic temporal interactions:

  • XIRP1 recruitment to cytosolic Listeria occurs in an ActA-dependent manner during bacterial replication and motility

  • Capturing these dynamic interactions requires precise temporal resolution

  • Conventional fixation methods may miss transient interactions

• Spatial resolution constraints:

  • While super-resolution SIM has visualized XIRP1 localization , limitations persist in:

    • Simultaneously resolving XIRP1 and multiple cytoskeletal proteins

    • Capturing the three-dimensional organization of XIRP1-cytoskeleton-bacteria complexes

    • Distinguishing between direct and indirect protein interactions

• Biochemical verification challenges:

  • Cell lysis can disrupt weak or transient cytoskeletal interactions

  • Difficulty separating bacterial and host protein complexes

  • Limited antibody compatibility for sequential immunoprecipitation

• Technical solutions being developed:

  • Photo-activatable proximity labeling to capture transient interactions

  • Live-cell super-resolution imaging with reduced phototoxicity

  • Correlative light and electron microscopy to link fluorescence patterns to ultrastructure

  • Improved co-immunoprecipitation protocols optimized for cytoskeletal proteins

These limitations require integrated approaches combining advanced imaging, biochemical analyses, and proximity labeling techniques to fully understand XIRP1's dynamic interactions during infection.

How does XIRP1 recruitment to Listeria surfaces correlate with actin-based motility phases?

XIRP1 recruitment to Listeria surfaces shows specific patterns that correlate with distinct phases of actin-based motility. Based on confocal and super-resolution microscopy studies :

The recruitment process follows a specific sequence:

• Initial recruitment requires:

  • Bacterial escape from the phagosome (hly plcAB mutants fail to recruit XIRP1)

  • Expression of the ActA protein (ΔactA mutants show no XIRP1 recruitment)

  • Presence of host XIRP1 (recruitment occurs in IFN-γ-activated macrophages)

• During bacterial replication:

  • Super-resolution microscopy shows XIRP1 localization around dividing bacteria

  • This precedes extensive actin tail formation

• During active motility:

  • XIRP1 remains associated with the bacterial surface and actin tail

  • Orthogonal views from SIM reveal distribution patterns that differ between motile and stationary bacteria

Functionally, XIRP1 appears to be co-opted by Listeria, as chromosomal removal of XIRP1 in mice impaired bacterial dissemination and increased resistance to infection . This suggests XIRP1 plays a critical role in facilitating bacterial escape from macrophages, particularly in the hostile environment of IFN-γ-activated cells.

What methodological approaches can resolve conflicting data regarding XIRP1's role in tumor suppression versus progression?

The search results present evidence that XIRP1 may function as a tumor suppressor in glioblastoma, with lower expression correlating with higher tumor grade and worse prognosis . To resolve potential conflicts regarding XIRP1's role across different cancer contexts, researchers should implement the following methodological approaches:

• Comprehensive tissue-specific analysis:

  • Tissue microarray analysis with quantitative H-score methodology across multiple tumor types

  • Single-cell RNA sequencing to identify cell-type specific expression patterns

  • Correlation of XIRP1 expression with hypoxic markers in different tumor regions

  • Stratification of samples by molecular subtypes and genetic backgrounds

• Functional validation through genetic manipulation:

  • CRISPR-Cas9 knockout studies in different cell types

  • Rescue experiments with different XIRP1 isoforms

  • Temporal control of XIRP1 expression using inducible systems

  • Domain-specific mutagenesis to isolate function-specific effects

• Mechanism dissection:

  • Analysis of XIRP1 interaction with regulatory proteins like HuR

  • Investigation of circRNA-mediated regulation (circPLOD2a/b)

  • Examination of transcriptional and post-transcriptional control mechanisms

• Controlled in vivo studies:

  • Orthotopic xenograft models with precisely controlled XIRP1 manipulation

  • Genetic mouse models with tissue-specific XIRP1 deletion

  • Patient-derived organoids to maintain tumor heterogeneity

  • Sequential sampling to track XIRP1 expression during tumor evolution

These approaches can distinguish between direct and indirect effects of XIRP1 on tumor biology and resolve seemingly conflicting data across different cancer types.

How can super-resolution microscopy enhance the study of XIRP1 dynamics in podosomes?

Super-resolution microscopy, particularly structured illumination microscopy (SIM), has already provided valuable insights into XIRP1 localization in macrophage podosomes . To further enhance understanding of XIRP1 dynamics:

• Multi-modal super-resolution approaches:

  • Combining SIM with STORM or PALM techniques would provide complementary spatial information

  • SIM offers excellent live-cell compatibility (100-120 nm resolution), while STORM/PALM provide higher static resolution (20-30 nm)

  • Correlative light and electron microscopy could link XIRP1 fluorescence patterns to ultrastructural features

• Advanced live-cell imaging methodologies:

  • Fluorescent protein fusions of XIRP1 compatible with live SIM imaging

  • Lattice light-sheet microscopy with SIM for extended 3D imaging with reduced phototoxicity

  • FRAP (Fluorescence Recovery After Photobleaching) combined with SIM to quantify XIRP1 turnover rates in podosomes

• Multi-color co-localization studies:

  • Three-dimensional, multi-color SIM to simultaneously visualize:

    • XIRP1 with podosome core components (actin, Arp2/3)

    • XIRP1 with podosome ring proteins (vinculin, talin)

    • XIRP1 with signaling molecules that regulate podosome dynamics

  • Quantitative co-localization analysis with nanometer precision

• Specialized applications for infection studies:

  • Simultaneous visualization of XIRP1, bacterial pathogens, and podosome components

  • Tracking XIRP1 redistribution from podosomes to bacterial surfaces during infection

  • Quantification of dynamic recruitment kinetics

These advanced microscopy approaches would provide unprecedented insights into the spatial organization, temporal dynamics, and functional significance of XIRP1 in macrophage podosomes and during host-pathogen interactions .