ARL4D Antibody

What is ARL4D and why is it important in immunological research?

ARL4D (ADP-ribosylation factor-like 4D) is a small GTPase belonging to the ARF/ARL protein family of Ras-related GTPases. It plays critical roles in:

• Membrane transport processes

• Actin cytoskeleton remodeling

• T-cell biology, particularly regulatory T-cell differentiation

• Cellular migration and adhesion

In immunological research, ARL4D is particularly significant because it restricts both IL-2 production and responsiveness to IL-2 in T cells, as measured by STAT5 phosphorylation . ARL4D-deficient CD4 T cells convert more efficiently into Foxp3+ induced T-regulatory cells (iTreg) in vitro in the presence of αCD3ε and TGFβ, associated with enhanced IL-2 secretion . This makes ARL4D antibodies essential tools for studying T-cell differentiation and immune regulation.

What cellular compartments does ARL4D localize to?

ARL4D exhibits a complex subcellular distribution pattern that is dependent on its activation state and post-translational modifications:

Endogenous ARL4D localizes predominantly to the plasma membrane and membrane fractions in biochemical fractionation studies . The GTP-dependent localization of ARL4D at the plasma membrane is critical for its function in recruiting cytohesin-2/ARNO, which subsequently activates ARF6 and modulates actin remodeling .

How is ARL4D expression regulated in T cells?

ARL4D mRNA expression is dynamically regulated during T-cell activation:

• In resting CD4 T cells, ARL4D is constitutively expressed

• Upon stimulation with αCD3ε alone or in combination with αCD28, ARL4D mRNA expression is rapidly but temporarily downregulated

• This downregulation correlates inversely with an increase in IL-2 mRNA and cytokine production, particularly evident at 48 hours post-stimulation

• ARL4D mRNA is upregulated after 48h compared to 24h, most efficiently after αCD3ε stimulation alone

This regulated expression pattern suggests that ARL4D functions as a checkpoint in T-cell activation, with its expression levels directly influencing IL-2 production and downstream T-cell differentiation pathways.

What controls should be included when validating an ARL4D antibody?

Proper validation of ARL4D antibodies requires multiple controls:

In particular, immunoblot antibody competition analysis using peptide immunogens is critical. In HeLa cells, detection of a 25-kDa protein was abolished by preincubation of the antibody with the ARL4D-B peptide immunogen but not with ARL4D-N peptide, demonstrating specificity .

How can I differentiate between active and inactive forms of ARL4D?

Distinguishing between active (GTP-bound) and inactive (GDP-bound) forms of ARL4D can be accomplished through several approaches:

• Mutant constructs as tools:

  • ARL4D(Q80L): A constitutively active mutant that mimics the GTP-bound state

  • ARL4D(T35N): An inactive mutant that mimics the GDP-bound state

  • ARL4D(G2A): A myristoylation-deficient mutant that cannot properly localize to membranes

• Interaction-based detection:

  • GTP-bound ARL4D (active) interacts with the C-terminal PH domain and polybasic c domain of ARNO

  • GST-ARNO pull-down assays can selectively capture active ARL4D forms

  • In coimmunoprecipitation experiments, ARNO only binds ARL4DWT, ARL4D(Q80L), or ARL4D(G2A), but not ARL4D(T35N)

• Subcellular localization analysis:

  • Active ARL4D concentrates at the plasma membrane, particularly in membrane ruffles

  • Inactive ARL4D shows more nuclear and perinuclear punctate distribution with reduced plasma membrane association

What are the key methodological considerations when using ARL4D antibodies for immunofluorescence?

Successful immunofluorescence with ARL4D antibodies requires careful attention to several parameters:

• Fixation method: Paraformaldehyde (typically 4%) preserves membrane structures where ARL4D localizes

• Antibody validation: Use competition assays with specific peptides to confirm signal specificity:

  • The signals of ARL4D were abolished when the antibody was preincubated with the ARL4D-B peptide

  • DMSO or ARL4D-N peptide preincubation did not affect detection

• Detection systems:

  • High-quality secondary antibodies (Alexa Fluor 594, 488, or 350-conjugated anti-rabbit antibodies)

  • Confocal microscopy for detailed subcellular localization

  • Z-stacking to capture complete membrane-associated signals

• Multiple markers analysis:

  • Co-staining with plasma membrane markers (e.g., PMCA) to confirm membrane localization

  • Nuclear counterstaining to assess nuclear distribution

How should I design experiments to study ARL4D's role in T-cell differentiation?

To effectively study ARL4D's role in T-cell differentiation, consider the following experimental design:

• T-cell isolation and activation protocols:

  • Isolate CD4 T cells using magnetic or flow cytometry-based methods

  • Stimulate with plate-bound anti-CD3ε with or without soluble anti-CD28

  • Monitor ARL4D expression kinetics using qPCR at multiple timepoints (24h, 48h)

• In vitro iTreg differentiation assay:

  • Culture CD4 T cells with αCD3ε and TGFβ

  • Compare wild-type and ARL4D-deficient cells for Foxp3 induction efficiency

  • Measure IL-2 production using ELISA

• Signaling analysis:

  • Assess STAT5 phosphorylation as a readout of IL-2 responsiveness

  • Compare pSTAT5+ cell frequencies between ARL4D-proficient and deficient cells

• Functional validation using animal models:

  • Use both global knockout and cell-specific knockout models

  • Consider transfer colitis models to assess the pathogenic potential of ARL4D-deficient T cells

How can I investigate the molecular mechanism between ARL4D, ARNO, and actin remodeling?

Investigating this molecular mechanism requires sophisticated approaches:

• Domain mapping studies:

  • The C-terminal 140 amino acids of ARNO (ARNOCT) are sufficient for interaction with ARL4D

  • Both the PH domain and polybasic c domain of ARNO are necessary for optimal interaction

  • The C-terminal basic amino acids in ARNO are not critical for ARL4D interaction

• Structure-function analysis:

  • ARL4D interaction with ARNO requires its C-terminal NLS domain

  • ARL4DΔC mutant fails to interact with ARNO in pull-down and co-immunoprecipitation assays

• Functional assays for actin remodeling:

  • ARL4D(Q80L) induces disassembly of actin stress fibers

  • This effect is blocked by expression of inactive ARNO(E156K) or siRNA knockdown of ARNO

  • These phenotypes can be quantified using phalloidin staining and image analysis

• ARF6 activation measurement:

  • ARL4D(Q80L) increases GTP-bound ARF6

  • Pull-down assays with ARF6 effector domains can quantify this activation

  • This establishes the pathway: ARL4D → ARNO recruitment → ARF6 activation → actin remodeling

What approaches can address contradictory results between different ARL4D antibodies?

When facing inconsistent results between different ARL4D antibodies, consider:

• Epitope mapping and antibody characterization:

  • Different antibodies may target distinct epitopes (e.g., a.a. 2-18 vs. a.a. 139-155)

  • Systematically compare antibodies raised against different regions

• Detection of alternative forms:

  • Be aware that a ~42-kDa band is sometimes detected in nuclear fractions

  • This band is abolished by specific ARL4D-B antigen, suggesting it may be an alternative form

• Subcellular fractionation verification:

  • Combine antibody detection with proper fractionation controls

  • For membrane fractions: Na+/K+ ATPase or PMCA

  • For nuclear fractions: appropriate nuclear markers

  • Endogenous ARL4D distributes mainly in the membrane fraction

• Combined methodological approach:

  • Integrate results from multiple techniques (Western blot, immunofluorescence)

  • Use genetic approaches (knockdown/knockout) for definitive validation

How can I design experiments to investigate the mechanistic link between ARL4D and IL-2 regulation?

To elucidate the mechanistic link between ARL4D and IL-2 regulation:

• Temporal analysis of expression:

  • The correlation between loss of ARL4D mRNA and increased IL-2 mRNA/protein is apparent, particularly at 48 hours post-stimulation

  • Compare different stimulation conditions (αCD3ε alone vs. αCD3ε/αCD28)

• Dose-response studies:

  • ARL4D-deficient CD4 T cells produce significantly more IL-2 particularly at lower αCD3ε concentrations

  • This effect appears to be IL-2-specific, as IFNγ production is not affected by ARL4D deficiency

• Genetic rescue experiments:

  • Reintroduce wild-type or mutant ARL4D into knockout cells

  • Determine which domains are essential for IL-2 regulation

• Integration with signaling pathways:

  • Investigate whether ARL4D affects IL-2 gene transcription or post-transcriptional regulation

  • Explore potential connections to TCR signaling pathways

What are the considerations for studying ARL4D in different cell types beyond T cells?

ARL4D functions in multiple cell types, requiring adaptation of research approaches:

When adapting ARL4D antibody-based studies to these diverse contexts, consider:

• Cell-specific expression patterns: Validate baseline expression and regulation

• Relevant functional readouts: Beyond actin remodeling, assess cell-type specific processes

• Co-expression with interacting partners: Verify presence of effectors like ARNO/cytohesin-2

How can I optimize ARL4D antibody-based co-immunoprecipitation experiments?

For successful co-immunoprecipitation of ARL4D with its binding partners:

• Buffer optimization:

  • Use buffers that preserve GTP-dependent interactions

  • Include appropriate detergents to solubilize membrane-associated ARL4D

• Experimental validation:

  • As demonstrated in the literature, ARL4D wild-type, ARL4D(Q80L), or ARL4D(G2A) can be co-immunoprecipitated with FLAG-ARNO

  • ARL4D(T35N) or ARL4DΔC fail to co-immunoprecipitate, confirming specificity

• Controls:

  • Include GTP-binding mutants (Q80L, T35N) to confirm nucleotide-dependence

  • Use domain mutants to verify interaction specificity

• Technical considerations:

  • Perform adequate cell lysis to release membrane-associated proteins

  • Consider crosslinking for transient interactions

  • Use GTPγS/GDP loading to stabilize specific nucleotide-bound states

What are the emerging applications of ARL4D antibodies in cancer research?

While early in development, ARL4D antibodies have potential applications in cancer research:

• Metastasis studies:

  • ARL4D's role in actin remodeling suggests involvement in cell migration

  • ARL4D-deficient cells show suppressed migration activity, similar to effects seen with cytohesin-2/ARNO or ARF6 depletion

• Signaling pathway analysis:

  • ARL4D's interaction with ARNO leads to ARF6 activation, which is implicated in cancer progression

  • Antibodies can help track these pathway activations in tumor tissues

• Tumor immune microenvironment:

  • ARL4D's role in T-cell biology suggests potential involvement in tumor immunity

  • Antibodies could help characterize T-cell states within tumors

How can I quantitatively assess ARL4D's impact on T-cell differentiation using antibody-based methods?

Quantitative assessment of ARL4D's impact on T-cell differentiation requires:

• Flow cytometry-based approaches:

  • Measure Foxp3 induction efficiency in ARL4D-proficient vs. deficient cells

  • Quantify pSTAT5+ cell frequencies as a readout of IL-2 responsiveness

  • Correlate with ARL4D protein levels

• Time-course analysis:

  • Track the temporal relationship between ARL4D downregulation and differentiation markers

  • Document key events in the differentiation timeline with antibody-based detection

• Multiplexed analysis:

  • Combine ARL4D detection with markers of T-cell subsets

  • Correlate ARL4D levels with functional outcomes across populations

ARL4D Antibody Epitope Information

ARL4D Mutant Constructs for Experimental Controls