ARPC5L Antibody

What is ARPC5L and what is its biological function?

ARPC5L (Actin-Related Protein 2/3 Complex Subunit 5-Like) is one of two isoforms of the ARPC5 subunit of the Arp2/3 complex, alongside ARPC5. The Arp2/3 complex plays a central role in actin polymerization dynamics. ARPC5L specifically drives nuclear actin polymerization upon T cell activation, while ARPC5 is primarily involved in cytoplasmic actin dynamics and TCR proximal signaling . ARPC5L plays crucial roles in cell motility, shape changes, migration, and invasion . The protein is predicted to enable actin filament binding activity and is involved in Arp2/3 complex-mediated actin nucleation .

What applications are validated for ARPC5L antibodies?

ARPC5L antibodies have been validated for several experimental applications:

The applications have been validated in multiple species including human, mouse, and rat samples .

How can I verify ARPC5L antibody specificity?

Testing antibody specificity is crucial due to the high similarity between ARPC5 and ARPC5L. Some commercially available ARPC5L antibodies may detect both isoforms. For example, research has shown that "the ARPC5L antibody also detects ARPC5 (marked by red asterisk)" . To verify specificity:

• Use known positive controls (U-87MG, LO2, HeLa cells, mouse brain, rat spleen)

• Include knockout or knockdown samples as negative controls

• Test in cell lines with verified expression of both ARPC5 and ARPC5L (e.g., Jurkat cells)

• Compare with the expression pattern observed in published research

The specificity of anti-ARPC5 and anti-ARPC5L antibodies has been confirmed using B16-F1 C5KO and C5LKO cell lines .

What is the typical expression profile of ARPC5L in different cell types?

ARPC5L expression shows significant cell type-specific and activation-dependent variation:

• In CD4 T cells: Expression is heterogeneous and correlates with activation status

  • Resting CD4 T cells: ~15% of cells express ARPC5L

  • Memory CD4 T cells: ~22% express ARPC5L

  • Activated CD4 T cells: ~31% express ARPC5L

  • Effector CD4 T cells: ~66% express ARPC5L

• Expressed in brain tissue (human, mouse, rat)

• Detected in various cell lines: U-87MG, LO2, HeLa, NIH/3T3

How can I distinguish between ARPC5 and ARPC5L in experimental samples?

Distinguishing between these two isoforms requires careful methodological considerations:

What methodological approaches are effective for studying ARPC5L's role in nuclear actin polymerization?

Research has established ARPC5L's specific role in nuclear actin polymerization, particularly in T cells. To study this function:

• CRISPR-Cas9 knockout models: Generate ARPC5L-specific knockout cell lines using ribonucleoprotein transfection approaches .

• Fluorescent tagging: Use mCherry-tagged ARPC5L to visualize subcellular localization during T cell activation .

• Activation assays: Implement T cell receptor (TCR) engagement protocols to trigger ARPC5L-dependent nuclear actin polymerization .

• Quantitative microscopy: Employ high-resolution imaging to quantify nuclear actin dynamics before and after activation.

• Complementation studies: Perform rescue experiments with wild-type or mutant ARPC5L constructs in knockout backgrounds to confirm functional specificity .

• Combined approaches: Integrate imaging with biochemical assays to correlate ARPC5L levels with nuclear actin polymerization: "While ARPC5L is heterogeneously expressed in individual CD4 T cells, it specifically drives nuclear actin polymerization upon T cell activation" .

How does ARPC5L expression affect cell migration and lamellipodial dynamics?

While ARPC5L drives nuclear actin polymerization, its sister isoform ARPC5 plays distinct roles in cytoplasmic actin dynamics and cell migration:

• Differential effects on lamellipodial morphology:

  • "Cells exclusively containing ArpC5L exhibited narrower and less protrusive lamellipodia"

  • "ArpC5-containing cells displayed even wider lamellipodia than wild-type (WT) cells"

• Impact on cellular motility:

  • ARPC5 (not ARPC5L) is "associated with enhanced cell motility and could be linked to increased cancer progression and metastasis"

• Experimental approaches for studying these differences:

  • Generate single and double knockout cell lines lacking either one or both ArpC5 isoforms

  • Implement multiscale approaches combining genetic engineering with structural biology

  • Analyze branch junction structures and resulting cytoskeletal architectures

  • Measure lamellipodial protrusion characteristics and actin network dynamics

What controls ARPC5L expression and how can researchers manipulate it experimentally?

ARPC5L expression is regulated in a complex manner, particularly in T cells:

• T cell activation regulation:

  • T cell activation significantly increases expression levels of Arp2/3 complex subunits

  • ARPC5L expression correlates with activation status, with highest expression in effector T cells

• Experimental induction:

  • TCR stimulation protocols can be used to induce ARPC5L expression

  • ARPC5L expression can be monitored at both mRNA (qRT-PCR) and protein (Western blot) levels

• Heterogeneous expression considerations:

  • ARPC5L shows heterogeneous expression in T cell populations

  • Single-cell analysis methods should be employed rather than bulk measurements

  • "Heterogeneously expressed molecules may thus be molecular discriminators that govern nuclear actin polymerization in CD4 T cells"

• Experimental manipulation:

  • CRISPR-Cas9 knockout approaches are effective for eliminating ARPC5L expression

  • Transgene expression systems can be used for controlled re-expression

  • Effects of knockout can be assessed by examining levels of other Arp2/3 complex subunits, which "remained largely unaffected but ARPC1B expression was reduced by almost 2-fold"

What are the best practices for using ARPC5L antibodies in immunofluorescence studies?

When using ARPC5L antibodies for immunofluorescence:

• Fixation protocols: Standard paraformaldehyde fixation (4%) is suitable for preserving ARPC5L epitopes.

• Subcellular localization patterns: Expect both diffuse cytoplasmic distribution and punctate staining in both cytoplasm and nucleus. Research shows that "both mCherry-tagged ARPC5 and ARPC5L had a diffuse cytoplasmic distribution but were also detected as punctae in the cytoplasm and the nucleus" .

• Activation-dependent changes: Upon T cell activation, particularly surface-mediated TCR engagement, enhanced staining at the plasma membrane may be observed .

• Specificity controls: Include appropriate knockout controls since "antibody staining did not allow to distinguish between the distribution of endogenous ARPC5 and ARPC5L" .

• Co-staining recommendations: Combine with markers for actin structures or T cell activation markers to correlate ARPC5L localization with functional outcomes.

How can I optimize Western blotting protocols for ARPC5L detection?

Optimizing Western blot protocols for reliable ARPC5L detection:

• Sample preparation considerations:

  • Ensure complete cell lysis to access both nuclear and cytoplasmic pools

  • Use phosphatase inhibitors if studying activated T cells

• Detection sensitivity:

  • ARPC5 may require "extended exposure" for detection

  • Verify detection at the expected molecular weight of 17kDa

• Antibody dilution optimization:

  • Start with recommended 1:500 - 1:2000 dilution range

  • Titrate to optimize signal-to-noise ratio for your specific samples

• Controls:

  • Include positive control samples (U-87MG, LO2, HeLa, mouse brain, rat spleen)

  • Where possible, include knockout samples as negative controls

• Isoform discrimination:

  • Be aware that "the ARPC5L antibody also detects ARPC5 (marked by red asterisk)"

  • Use knockout samples of each isoform for definitive identification

What experimental approaches can reveal ARPC5L's role in T cell function beyond nuclear actin polymerization?

To investigate broader ARPC5L functions in T cells:

• Transcriptional regulation studies:

  • Compare gene expression profiles between ARPC5L knockout and control T cells

  • Focus on activation-dependent genes that may be regulated by nuclear actin dynamics

• Functional T cell assays:

  • Measure cytokine production capacity in ARPC5L knockout T cells

  • Assess T cell activation markers and proliferation responses

• Proximal signaling analysis:

  • Compare with ARPC5 knockout effects: "loss of ARPC5L had no effect on the amount of TCR signaling-induced microclusters formed or their pSLP-76 content"

  • Measure TCR signaling using phosphotyrosine analysis: "pTyr intensity in these microclusters was reduced relative to that in control cells, albeit to significantly lower extend than in C5 KO cells"

• Integrative approaches:

  • Combine imaging, functional, and biochemical approaches

  • Correlate ARPC5L expression levels with functional outcomes at the single-cell level

How can I address potential cross-reactivity issues with ARPC5L antibodies?

Cross-reactivity between ARPC5 and ARPC5L antibodies is a documented challenge:

• Validation in knockout models:

  • Confirm antibody specificity using ARPC5 and ARPC5L knockout cell lines

  • Research has utilized "B16-F1 C5KO and C5LKO lines" for antibody validation

• Epitope considerations:

  • Select antibodies raised against divergent regions between the isoforms

  • Consider using antibodies generated against recombinant fusion proteins containing specific sequences (e.g., "amino acids 1-100 of human ARPC5L (NP_112240.1)")

• Complementary approaches:

  • Use mRNA detection methods (qRT-PCR, RNA-FISH) in parallel with protein detection

  • Implement isoform-specific tagging strategies when overexpressing these proteins

• Specificity testing protocol:

  • Test antibodies against known positive samples expressing both isoforms

  • Include appropriate negative controls (knockout samples or tissues known to lack expression)

  • Document cross-reactivity patterns for accurate data interpretation

What methodological considerations are important when studying ARPC5L in cancer research?

ARPC5L's potential role in cancer progression requires specific experimental approaches:

• Expression analysis in cancer tissues:

  • Compare ARPC5L expression between normal and malignant tissues

  • Assess correlation with invasive and metastatic phenotypes

  • Note that "specifically ArpC5 over ArpC5L is associated with enhanced cell motility and could be linked to increased cancer progression and metastasis"

• Functional studies:

  • Generate ARPC5L knockout in cancer cell lines using CRISPR-Cas9

  • Assess effects on migration, invasion, and metastatic potential

  • Compare with ARPC5 knockout phenotypes to distinguish isoform-specific effects

• Nuclear function investigations:

  • Explore nuclear actin dynamics in cancer cells with altered ARPC5L expression

  • Investigate potential roles in gene expression regulation

• Translational relevance:

  • Correlate ARPC5L expression with clinical outcomes in cancer patients

  • Evaluate potential as a biomarker or therapeutic target

How can single-cell approaches enhance our understanding of ARPC5L biology?

Single-cell methods are particularly valuable for studying ARPC5L given its heterogeneous expression:

• Single-cell RNA sequencing applications:

  • Identify cellular subpopulations with differential ARPC5L expression

  • Research has shown that "ARPC5L displays the highest heterogenic expression within a given T cell population"

  • Map ARPC5L expression to specific cell states (e.g., activation, differentiation)

• Methodological implementation:

  • Use established single-cell RNA-seq protocols (e.g., 10x Genomics, Smart-seq2)

  • Apply UMAP or tSNE clustering to identify cell populations with variable ARPC5L expression

  • Correlate with expression of other genes involved in actin regulation

• Functional correlations:

  • Link ARPC5L expression to functional outcomes at the single-cell level

  • Research has shown that "the frequency of cells that express ARPC5L is similar to that of cells displaying nuclear actin polymerization in response to T cell activation"

• Integration with imaging:

  • Combine single-cell transcriptomics with imaging approaches to correlate mRNA expression with protein localization and function

  • Implement RNA-FISH for visualization of ARPC5L transcripts at the single-cell level

What emerging technologies might advance ARPC5L research?

Several cutting-edge approaches could significantly enhance our understanding of ARPC5L biology:

• Live-cell nuclear actin imaging:

  • Develop improved fluorescent probes for visualizing nuclear actin dynamics in real-time

  • Correlate with ARPC5L localization and function during T cell activation

• Proximity labeling approaches:

  • Implement BioID or APEX2 proximity labeling with ARPC5L as the bait protein

  • Identify context-specific interaction partners in nuclear versus cytoplasmic compartments

• Cryo-electron microscopy:

  • Determine high-resolution structures of ARPC5L-containing Arp2/3 complexes

  • Compare with ARPC5-containing complexes to identify structural differences that explain functional divergence

• Optogenetic manipulation:

  • Develop tools for spatiotemporal control of ARPC5L activity

  • Dissect compartment-specific functions through targeted activation/inhibition

• CRISPR-based screening:

  • Implement genome-wide CRISPR screens to identify synthetic lethal interactions with ARPC5L

  • Discover new regulatory pathways that control ARPC5L expression and function

These advanced approaches will help resolve outstanding questions about the unique roles of ARPC5L in nuclear actin polymerization and its broader implications for cell biology.