ABLIM1 Antibody

What is ABLIM1 and what cellular functions does it perform?

ABLIM1 (actin-binding LIM protein 1) is a member of the LIM-domain protein family that mediates interactions between actin filaments and cytoplasmic targets. It functions as a non-erythroid cell-specific cortex organizer, governing the formation of dense interconnected cortical actin meshwork that prevents mechanical tension-induced blebbing during cellular activities such as spreading and migration. In-vitro assays demonstrate that ABLIM1 can crosslink and bundle F-actin to induce dense F-actin network formation . Recent research has also identified ABLIM1 as having ubiquitin E3 ligase activity, expanding our understanding of its cellular functions beyond cytoskeletal organization .

Where is ABLIM1 typically localized within cells?

Immunostaining has revealed that ABLIM1 is highly enriched at cell edges in RPE1 and U2OS cells, where its immunofluorescent signals colocalize with those of βII spectrin, a known cell cortex marker. When cells are partially detached from the substratum and become rounded, ABLIM1 is clearly visible at the cortex, appearing more punctate than βII spectrin . This cortical localization aligns with its role in actin cytoskeleton organization.

What are the validated applications for ABLIM1 antibody?

ABLIM1 antibody has been validated for multiple applications including:

• Western Blot (WB)

• Immunohistochemistry (IHC)

• Immunofluorescence (IF-P) for paraffin-embedded tissues

• Immunofluorescence (IF/ICC) for cells

• ELISA

The antibody shows reactivity with human, mouse, and rat samples, making it valuable for comparative studies across different model organisms .

What are the recommended dilutions for different applications of ABLIM1 antibody?

The recommended dilutions for the 15129-1-AP ABLIM1 antibody are as follows:

It is important to note that these are general recommendations, and the antibody should be titrated in each specific testing system to obtain optimal results as the ideal dilution may be sample-dependent .

What cell lines or tissues are suitable positive controls for ABLIM1 antibody validation?

Based on experimental data, the following samples can serve as positive controls:

For immunohistochemistry applications, antigen retrieval with TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may be used as an alternative .

How is ABLIM1 expression altered in cancer, and does it have a consistent role across different cancer types?

ABLIM1 exhibits context-dependent roles in cancer pathology. In glioblastoma (GBM), ABLIM1 functions as a tumor suppressor, with lower mRNA levels observed in GBM compared to other glioma types or normal brain tissues. Higher ABLIM1 protein expression correlates with smaller tumor size and better cancer-specific survival in GBM patients. Functionally, ABLIM1 overexpression significantly inhibits U87 and U251 glioblastoma cell proliferation and colony formation, with consistent tumor-suppressing effects observed in mouse models .

Conversely, in colorectal cancer (CRC), ABLIM1 appears to promote tumor growth. Research indicates that ABLIM1 is upregulated in CRC patients, with high expression levels predicting shorter disease-free survival time. Knockdown of ABLIM1 in CRC cell lines leads to inhibited cell proliferation, migration, and invasion capabilities in vitro and impaired growth of tumor xenografts and liver metastasis lesions in vivo .

These opposing roles highlight the importance of tissue-specific context when studying ABLIM1's functions in cancer.

Is ABLIM1 implicated in non-cancer pathologies?

ABLIM1 has been implicated in interstitial lung diseases (ILDs). Notably, many ILD patients have circulating antibodies against ABLIM1, which is specifically upregulated in structural cells of fibrotic lungs. This autoantibody response to ABLIM1 correlates with disease parameters, suggesting ABLIM1 may be part of a common immunopathological mechanism in pulmonary fibrosis . This evidence establishes ABLIM1 as a potential novel autoantigen and biomarker in fibrotic lung conditions.

How does ABLIM1 contribute to the NF-κB signaling pathway?

Recent research has identified ABLIM1 as a novel ubiquitin E3 ligase that can interact with components of the NF-κB pathway. Studies in colorectal cancer cells have examined ABLIM1's relationship with IκBα and p65 (RelA), core components of the NF-κB signaling pathway. Experimental approaches using immunoblotting for phosphorylated IκBα (p-IκBα S36) and phosphorylated p65 (p-p65 S536), along with immunofluorescence to track p65 nuclear translocation, have been employed to elucidate ABLIM1's role in this pathway . This emerging research area suggests ABLIM1 may influence inflammatory and cell survival pathways through its E3 ligase activity.

How can I validate the specificity of ABLIM1 antibody in my experimental system?

To rigorously validate ABLIM1 antibody specificity, employ these complementary approaches:

• Pre-incubation specificity test: Pre-incubate the anti-ABLIM1 antibody with purified His-tagged human ABLIM1, related proteins (e.g., ABLIM3), and unrelated proteins (e.g., GFP). Only pre-incubation with His-ABLIM1 should abolish the immunofluorescent signals .

• RNAi validation: Deplete ABLIM1 using specific siRNAs (such as abL1-i1) and confirm loss of antibody signal through immunofluorescence or Western blotting .

• RNAi-resistant rescue: Create an RNAi-resistant isoform of ABLIM1 by mutating the target sequence of siRNA without altering the coding amino acids (e.g., abLIM1R). Expression of this construct in cells depleted of endogenous ABLIM1 should restore antibody signal, confirming specificity .

These complementary approaches provide strong evidence for antibody specificity when consistent results are obtained across methods.

What are effective approaches for studying ABLIM1's role in actin cytoskeleton dynamics?

To investigate ABLIM1's functions in actin cytoskeleton organization:

• Depletion and phenotype analysis: Use RNAi to deplete ABLIM1, then examine cell morphology and actin cytoskeleton organization using phalloidin staining. ABLIM1-depleted cells exhibit distinct phenotypes, including lack of dense interwoven cortical actin meshwork and presence of long cortical actin bundles along the cell axis .

• Blebbing assays: Assess mechanical tension-induced blebbing during cell spreading after ABLIM1 depletion. Modulate cortical tensions by culturing cells to confluency or inhibiting myosin activity to determine if the blebbing phenotype can be suppressed .

• Rescue experiments: Reintroduce wild-type or mutant ABLIM1 constructs to determine which domains are essential for actin organization functions.

• In vitro actin organization assays: Purify ABLIM1 protein and assess its ability to crosslink and bundle F-actin in cell-free systems to directly characterize its biochemical activity on actin filaments .

What methodologies can be used to investigate ABLIM1's ubiquitin E3 ligase activity?

To investigate ABLIM1's recently identified E3 ligase activity:

• Protein expression systems: Generate stable cell lines overexpressing ABLIM1 with epitope tags (e.g., Flag-HA-ABLIM1) or use transient transfection of ABLIM1 constructs into appropriate cell lines such as HCT116, SW620, RKO, or 293T cells .

• Ubiquitination assays: Transfect cells with Flag-tagged RELA (p65) and His-tagged ubiquitin along with ABLIM1 constructs to assess ubiquitination of potential substrates. Perform immunoprecipitation followed by immunoblotting with anti-ubiquitin antibodies to detect ubiquitinated proteins .

• Subcellular localization analysis: Use immunofluorescence with specific antibodies against ABLIM1 and potential substrates (e.g., p65/RelA) to track changes in subcellular localization (nuclear versus cytoplasmic) following ABLIM1 manipulation .

• Functional assays: Measure downstream effects of NF-κB pathway modulation, such as expression of target genes like CCL20, following ABLIM1 overexpression or knockdown .

How can I reconcile differing observations about ABLIM1's molecular weight?

The discrepancy between ABLIM1's calculated molecular weight (88 kDa) and observed bands (48-52 kDa and 105-110 kDa) may be explained by several factors that warrant investigation:

• Alternative splicing: ABLIM1 may exist in multiple isoforms due to alternative splicing. Verify which isoforms are expressed in your experimental system using isoform-specific primers for RT-PCR or antibodies that recognize specific regions.

• Post-translational modifications: ABLIM1 may undergo modifications like phosphorylation, ubiquitination, or proteolytic processing. Employ phosphatase treatment, deubiquitinating enzymes, or protease inhibitors to determine if these modifications contribute to altered migration patterns.

• Protein complexes: The higher molecular weight band (105-110 kDa) may represent ABLIM1 in stable protein complexes resistant to SDS-PAGE denaturation. Use more stringent sample preparation conditions or native gel electrophoresis to investigate this possibility.

• Cross-reactivity: Validate that all observed bands are specific to ABLIM1 using ABLIM1 knockdown or knockout samples as negative controls.

What experimental approaches can resolve contradictory findings about ABLIM1's role in different cancers?

To address the opposing roles of ABLIM1 in glioblastoma (tumor suppressor) versus colorectal cancer (oncogenic) :

• Tissue-specific interactome analysis: Identify ABLIM1-interacting partners in different cancer types using co-immunoprecipitation followed by mass spectrometry to determine if ABLIM1 engages with different protein networks in a tissue-specific manner.

• Domain-specific functions: Generate constructs expressing specific ABLIM1 domains to determine if different functional regions predominate in different cancer contexts.

• Signaling pathway analysis: Systematically investigate ABLIM1's effects on key oncogenic pathways (MAPK/ERK, PI3K/AKT, NF-κB, Wnt/β-catenin) across cancer types to identify context-dependent regulation.

• Epigenetic and mutation analysis: Examine if ABLIM1 gene mutations, promoter methylation, or histone modifications differ between cancer types, potentially explaining functional differences.

• In vivo models: Employ tissue-specific ABLIM1 knockout or overexpression in multiple cancer models to directly compare effects across cancer types.

What are the best approaches to identify and validate novel ABLIM1 substrates as an E3 ligase?

To systematically identify and validate substrates of ABLIM1's E3 ligase activity:

• Global ubiquitinome analysis: Perform quantitative proteomics comparing ubiquitinated proteins in control versus ABLIM1-depleted or overexpressing cells. Use diGly remnant antibodies to enrich ubiquitinated peptides for mass spectrometry analysis.

• Proximity-based labeling: Employ BioID or TurboID approaches with ABLIM1 fusion proteins to identify proteins in close proximity that may be potential substrates.

• In vitro ubiquitination assays: Use purified components (E1, E2, ABLIM1, and candidate substrates) to reconstitute ubiquitination reactions in vitro, followed by immunoblotting to detect ubiquitin conjugation.

• Ubiquitination site mapping: For validated substrates, identify specific lysine residues targeted by ABLIM1 through site-directed mutagenesis of candidate sites followed by ubiquitination assays.

• Functional validation: Determine if ubiquitination by ABLIM1 affects substrate stability, localization, or activity through phenotypic rescue experiments using ubiquitination-resistant substrate mutants.

What are common issues when using ABLIM1 antibody for immunohistochemistry and how can they be resolved?

Common challenges and their solutions include:

• Weak or variable signal:

  • For ABLIM1 IHC, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 can be used as an alternative

  • Optimize antibody concentration (start with 1:20-1:200 dilution range)

  • Extend primary antibody incubation (overnight at 4°C is often effective)

  • Use amplification systems like tyramide signal amplification if signal remains weak

• High background:

  • Increase blocking time or blocking agent concentration

  • Include additional washing steps with higher stringency buffers

  • If using DAB detection, reduce development time

  • Pre-absorb antibody with blocking peptides

• Non-specific staining:

  • Include appropriate negative controls (no primary antibody, isotype control)

  • Validate specificity with ABLIM1-depleted samples

  • Consider using monoclonal antibodies if polyclonal shows cross-reactivity

• Inconsistent results between experiments:

  • Standardize fixation protocols (time, temperature, fixative composition)

  • Use automated staining platforms when available

  • Prepare larger batches of working antibody dilutions

How should researchers interpret discrepancies between ABLIM1 protein and mRNA expression?

When investigating discrepancies between ABLIM1 protein and mRNA levels:

• Technical considerations:

  • Verify antibody specificity using the validation methods described in section 4.1

  • Confirm primer specificity for ABLIM1 mRNA detection, considering potential isoforms

  • Use multiple reference genes for qRT-PCR normalization

• Biological explanations:

  • Post-transcriptional regulation: Investigate microRNA targeting of ABLIM1 mRNA

  • Protein stability: Assess ABLIM1 protein half-life using cycloheximide chase experiments

  • Tissue-specific translational control: Compare polysome-associated ABLIM1 mRNA across tissues

• Experimental approach:

  • Perform parallel analysis of ABLIM1 mRNA and protein from the same samples

  • Use reporter constructs containing ABLIM1 UTRs to identify regulatory elements

  • Consider single-cell analysis to detect population heterogeneity that might be masked in bulk measurements

What controls should be included when studying ABLIM1 knockdown or overexpression phenotypes?

To ensure robust and reproducible results when manipulating ABLIM1 expression:

• Essential knockdown controls:

  • Multiple independent siRNAs/shRNAs targeting different regions of ABLIM1 to minimize off-target effects

  • Non-targeting scramble control siRNA/shRNA with similar GC content

  • RNAi-resistant ABLIM1 rescue construct to confirm phenotype specificity

  • Quantification of knockdown efficiency at both mRNA and protein levels

• Overexpression controls:

  • Empty vector expressing the same tag as the ABLIM1 construct

  • Expression level verification by Western blot and qRT-PCR

  • Subcellular localization confirmation by immunofluorescence

  • Domain mutants to identify functional regions responsible for observed phenotypes

• Experimental validation:

  • Complementary approaches (e.g., CRISPR/Cas9 knockout, dominant-negative constructs)

  • Dose-response assessments for phenotypes relating to varying levels of ABLIM1 expression

  • Parallel experiments in multiple cell lines to determine context-specificity

  • In vivo validation when possible (e.g., tissue-specific knockout or overexpression)

By incorporating these comprehensive controls, researchers can minimize misinterpretation and establish causality between ABLIM1 manipulation and observed phenotypes.