CARMIL1 Antibody

What is CARMIL1 and why is it important in cellular processes?

CARMIL1 (Capping protein, Arp2/3, and Myosin-I Linker protein 1) is a large, multidomain, membrane-associated protein that regulates actin assembly and Rho-family GTPases . It contributes significantly to the dendritic actin network assembly of lamellipodia and ruffles, functioning upstream of Rac1 signaling pathways . Recent research has revealed CARMIL1's essential role in inflammatory signaling, particularly in IL-1 receptor pathways .

For experimental characterization, immunofluorescence microscopy combined with functional assays (cell migration, focal adhesion formation) provides comprehensive insights into CARMIL1's cellular roles.

What are the known isoforms of CARMIL1 and how do they differ structurally?

Research has identified at least two CARMIL1 isoforms:

• CARMIL1a: A longer form containing an insertion of 135 bases between positions 3906 and 3907 compared to the shorter form

• CARMIL1b: The shorter form predicted by NCBI sequence NM_017640

These isoforms may have distinct functional properties, though most published research has focused on CARMIL1a . When designing experiments, researchers should consider which isoform they're targeting and whether their antibodies can distinguish between these variants.

How should researchers validate CARMIL1 antibody specificity?

A multi-tiered validation approach is essential:

• Western blot analysis comparing wild-type versus CARMIL1 knockout (CRISPR-Cas9) or knockdown (siRNA) cells

• Immunoprecipitation followed by mass spectrometry to confirm pulled-down proteins

• Immunofluorescence comparing staining patterns in control versus CARMIL1-depleted cells

• Testing antibody recognition of recombinant CARMIL1 fragments

Chicken antibodies against human CARMIL1 fragment (538-1371) have been successfully used in previous studies , providing a benchmark for validation.

What are optimal conditions for CARMIL1 immunoprecipitation experiments?

For effective co-immunoprecipitation of CARMIL1 and binding partners:

• Cell lysis buffer composition: 20 mM Tris/HCl (pH 7.5), 100 mM NaCl, 0.5% NP-40, 10% glycerol, and protease inhibitor cocktail

• Centrifugation: Remove cellular debris before immunoprecipitation

• Antibody incubation: 2 hours at 4°C with Dynabeads precoated with anti-GFP (for GFP-tagged CARMIL1) or anti-FLAG M2 affinity beads (for FLAG-tagged CARMIL1)

• Washing: Three washes with lysis buffer

• Elution: Boil with 2× SDS loading buffer before SDS-PAGE and immunoblotting

For stimulus-dependent interactions, compare unstimulated versus IL-1-treated conditions, as IL-1 significantly enhances CARMIL1 association with IL-1R1 and IRAK .

How can researchers assess CARMIL1's role in inflammatory signaling pathways?

Time-course experiments are recommended to capture the dynamics of these responses, with measurements at multiple timepoints after IL-1 stimulation (typically 0, 15, 30 minutes for signaling; 6 and 24 hours for gene expression) .

What techniques can reveal CARMIL1 localization during cellular responses?

For optimal immunofluorescence localization:

• Focal adhesion enrichment: Use collagen-coated bead preparations to isolate focal adhesion-associated proteins, where CARMIL1 is enriched following IL-1 stimulation

• Leading edge localization: Co-stain for Arp2/3, cortactin, or VASP to visualize CARMIL1 at lamellipodial leading edges

• Cytoskeletal connections: Examine CARMIL1 co-localization with actin filament-enriched extensions involved in ECM degradation

• Controls: Include paxillin as a positive control for focal adhesion enrichment and GAPDH as a negative control for cytosolic contamination

Image analysis should quantify colocalization coefficients and enrichment ratios relative to cytoplasmic signals.

How does the leucine-rich repeat (LRR) domain of CARMIL1 regulate IL-1 signaling?

The LRR region of CARMIL1 plays a crucial regulatory role in inflammatory pathways:

• Direct associations: The LRR domain associates with IL-1 receptor type 1 (IL-1R1) and IL-1 receptor-associated kinase (IRAK)

• Functional impact: Cells expressing CARMIL1 mutants lacking the LRR domain show approximately 45% lower ERK activation and 40% reduced MMP3 expression

• Therapeutic potential: Cell-permeable TAT CARMIL1 peptides that compete with IL-1R1 and IRAK binding to the LRR reduce collagen degradation by 43%

These findings position the LRR domain as a critical mediator between cytoskeletal regulation and inflammatory signaling, suggesting potential therapeutic applications for inflammatory conditions.

What distinguishes CARMIL1 from other CARMIL family members in cellular function?

CARMIL1 and CARMIL2 (also known as RLTPR) exhibit important functional differences:

This functional divergence highlights the importance of isoform-specific approaches when targeting CARMIL proteins in research or therapeutic development.

How can researchers manipulate CARMIL1 function to study its role in disease models?

Multiple approaches for functional manipulation include:

• Genetic approaches:

  • CRISPR-Cas9 knockout systems have successfully generated CARMIL1-null cell lines

  • siRNA knockdown targeting specific CARMIL1 regions

  • Expression of domain-specific mutants (particularly LRR deletion mutants)

• Peptide inhibitors:

  • Cell-permeable TAT CARMIL1 peptides that compete with binding partners

  • These peptides have shown efficacy in reducing collagen degradation by 43% in fibroblasts

• Experimental readouts:

  • ERK phosphorylation (reduced by ~72% in CARMIL1 KO cells)

  • MMP3 expression (reduced by ~40%)

  • Focal adhesion formation and actin-filament-enriched extensions

What are common challenges when working with CARMIL1 antibodies?

Always include both positive controls (known CARMIL1-expressing cells) and negative controls (CARMIL1 knockout cells) to establish assay validity .

How can researchers address discrepancies between antibody-based results and functional data?

When antibody results conflict with functional observations:

• Verify antibody specificity using multiple validation approaches

• Consider epitope accessibility – protein interactions may mask antibody binding sites

• Evaluate isoform specificity – determine if your antibody recognizes all relevant CARMIL1 isoforms

• Test alternative fixation and permeabilization protocols for immunofluorescence

• Implement orthogonal techniques (fluorescent protein tagging, mRNA quantification)

For comprehensive validation, combine biochemical approaches (immunoprecipitation, Western blotting) with cellular techniques (immunofluorescence, live imaging) .

What controls are essential when studying CARMIL1-protein interactions?

For robust interaction studies:

• Negative controls:

  • Immunoprecipitation with non-specific IgG (shown to yield minimal signal compared to specific antibodies)

  • CARMIL1 knockout or knockdown cells

  • Unstimulated conditions (for stimulus-dependent interactions)

• Positive controls:

  • Known CARMIL1 interacting proteins (e.g., myosin 1E)

  • Tagged CARMIL1 constructs

• Validation strategies:

  • Reciprocal co-immunoprecipitation

  • Domain mapping using truncation mutants

  • Stimulus-response experiments (e.g., IL-1 treatment enhances CARMIL1-IL-1R1 interaction)

How is CARMIL1 being investigated as a potential therapeutic target?

CARMIL1's involvement in inflammatory signaling presents promising therapeutic applications:

• Current evidence:

  • The LRR region regulates IL-1 signaling through association with IL-1R1 and IRAK

  • Cell-permeable TAT CARMIL1 peptides reduce collagen degradation by 43% in fibroblasts

  • CARMIL1 knockout reduces IL-1-induced ERK activation by 72% and MMP3 expression by 40%

• Therapeutic strategies under investigation:

  • Peptide inhibitors targeting the LRR domain

  • Small molecule disruptors of CARMIL1-IL-1R1/IRAK interaction

  • Domain-specific inhibitory approaches

These approaches aim to modulate inflammatory responses while minimizing impact on essential cytoskeletal functions.

What techniques are emerging for studying CARMIL1 dynamics in live cells?

Advanced imaging approaches include:

• Live-cell imaging with fluorescently tagged CARMIL1

• Super-resolution microscopy to visualize CARMIL1 within cytoskeletal structures

• FRET-based biosensors to monitor CARMIL1 protein interactions

• Correlative light and electron microscopy for ultrastructural localization

• Optogenetic tools to spatiotemporally control CARMIL1 function

These techniques enable researchers to observe CARMIL1's dynamic behavior during processes like cell migration, inflammatory responses, and cytoskeletal remodeling with unprecedented resolution and temporal precision.

How might post-translational modifications regulate CARMIL1 function?

While specific post-translational modifications of CARMIL1 remain largely unexplored, research should consider:

• Potential phosphorylation sites that might regulate:

  • Interaction with binding partners (IL-1R1, IRAK)

  • Localization to specific cellular compartments

  • Conformational changes affecting function

• Methodological approaches:

  • Phosphoproteomic analysis comparing CARMIL1 modification states before/after stimulation

  • Site-directed mutagenesis of potential regulatory residues

  • Phospho-specific antibodies for tracking modification-dependent events

This represents a significant knowledge gap and opportunity for future research in CARMIL1 biology.