ASB2 Antibody

What is ASB2 and why is it important for research?

ASB2 (Ankyrin Repeat and SOCS Box-Containing 2) is a protein that functions as the specificity subunit of an E3 ubiquitin ligase complex. In humans, the canonical protein has 635 amino acid residues with a molecular mass of 70.2 kDa and is primarily localized in the cytoplasm . ASB2 is a member of the Ankyrin SOCS box (ASB) protein family, which plays crucial roles in cytoskeleton organization and differentiation processes . Alternative splicing produces two main isoforms: ASB2α, predominantly expressed in hematopoietic cells, and ASB2β, mainly expressed in muscle tissues . The importance of ASB2 in research stems from its involvement in critical cellular processes including protein degradation pathways, muscle differentiation, and hematopoietic cell development . Understanding ASB2 function contributes to our knowledge of fundamental cellular mechanisms and potential therapeutic applications in diseases where these pathways are dysregulated.

How do ASB2α and ASB2β isoforms differ structurally and functionally?

ASB2α and ASB2β differ in several key aspects:

Structural differences:

• ASB2α has a predicted molecular weight of 64 kDa, while ASB2β is larger at approximately 70 kDa

• Both isoforms retain the core ankyrin repeats and SOCS box domains that are characteristic of the ASB protein family

• ASB2β possesses a unique N-terminal extension that contains a ubiquitin-interacting motif (UIM) not present in ASB2α

• Both isoforms contain a BC-box that defines binding sites for the elongin BC complex and a Cul5 box that determines binding specificity for Cullin5

Functional differences:

• ASB2α is primarily expressed in hematopoietic cells and is involved in their differentiation

• ASB2β is predominantly expressed in muscle cells (skeletal, cardiac, and smooth muscle) and plays a critical role in muscle differentiation

• ASB2α targets both filamin A (FLNa) and filamin B (FLNb) for degradation

• ASB2β specifically targets filamin B (FLNb) but not filamin A for proteasomal degradation

• ASB2β expression is induced during myogenic differentiation and its knockdown delays processes such as myoblast fusion and expression of muscle contractile proteins

These differences highlight the tissue-specific roles of ASB2 isoforms in regulating cellular differentiation through selective protein degradation pathways.

What applications are ASB2 antibodies most commonly used for?

ASB2 antibodies are employed in various research applications, with their utility dependent on the specific antibody characteristics. The most common applications include:

• Western Blotting (WB): Widely used for detecting and quantifying ASB2 protein expression in cell or tissue lysates. Many commercial ASB2 antibodies are validated for WB, allowing researchers to distinguish between the α (64 kDa) and β (70 kDa) isoforms .

• Immunohistochemistry (IHC): Used to visualize ASB2 protein expression and localization in tissue sections. This application helps researchers understand the spatial distribution of ASB2 isoforms in different tissues, particularly in muscle and hematopoietic tissues .

• Immunofluorescence (IF): Allows for subcellular localization studies of ASB2, particularly its cytoplasmic distribution and potential co-localization with target proteins such as filamin .

• Enzyme-Linked Immunosorbent Assay (ELISA): Used for quantitative detection of ASB2 in solution, though less commonly than the imaging-based techniques .

• Fluorescence-Linked Immunosorbent Assay (FLISA): A specialized application combining fluorescence detection with immunosorbent assay principles, available with certain ASB2 antibodies .

• Protein Purification: Some ASB2 antibodies are purified through protein A columns followed by peptide affinity purification, making them suitable for isolating ASB2 protein complexes from cellular extracts .

When selecting an ASB2 antibody, researchers should consider the specific isoform they wish to detect (α or β), the species reactivity needed (human, mouse, rat), and whether they require an unconjugated antibody or one labeled with a detection tag (such as biotin or fluorophores) .

How can I validate the specificity of an ASB2 antibody?

Validating the specificity of an ASB2 antibody is crucial for experimental reliability. Here is a methodological approach to antibody validation:

• Western Blot Analysis with Recombinant Proteins:

  • Express tagged versions (e.g., Flag-tagged) of ASB2α and ASB2β in a heterologous system such as HeLa cells

  • Run protein samples on SDS-PAGE alongside negative controls (untransfected cells)

  • Perform western blotting with your ASB2 antibody

  • Confirm detection of bands at the expected molecular weights (64 kDa for ASB2α and 70 kDa for ASB2β)

  • Compare with detection using an anti-tag antibody to verify specificity

• Isoform-Specific Validation:

  • For antibodies claiming to detect specific isoforms (like the 2PNAB1 serum that specifically recognizes ASB2β), test against both isoforms to confirm selective detection

  • Antibodies targeting common regions (like the 1PLA serum directed against the C-terminus) should detect both isoforms

• Knockdown/Knockout Controls:

  • Generate ASB2 knockdown cells using shRNAs or siRNAs

  • Compare antibody signal between wild-type and knockdown samples

  • A specific antibody will show decreased signal intensity corresponding to the degree of knockdown

• Tissue Expression Pattern Analysis:

  • Test the antibody on tissues known to differentially express ASB2 isoforms (e.g., hematopoietic cells for ASB2α, muscle cells for ASB2β)

  • Confirm that detection patterns match known expression profiles

• Immunoprecipitation Followed by Mass Spectrometry:

  • Perform immunoprecipitation with the ASB2 antibody

  • Analyze pulled-down proteins by mass spectrometry

  • Confirm the presence of ASB2 and known interacting partners (e.g., elongin BC complex proteins, Cullin5, Rbx2)

• Cross-Reactivity Assessment:

  • Test the antibody against closely related proteins in the ASB family

  • Ensure the antibody does not cross-react with other family members

By following these validation steps, researchers can establish confidence in the specificity of their ASB2 antibody before proceeding with experimental applications.

How can ASB2 antibodies be used to study E3 ubiquitin ligase activity?

ASB2 antibodies are powerful tools for investigating the E3 ubiquitin ligase activity of ASB2-containing complexes through several sophisticated approaches:

• Immunoprecipitation-Based Ubiquitination Assays:

  • Immunoprecipitate ASB2 complexes from cell lysates using specific antibodies

  • Perform in vitro ubiquitination assays by adding recombinant E1, E2 (UbcH5a), ATP, and ubiquitin

  • Analyze formation of polyubiquitin chains by western blotting

  • Compare wild-type ASB2 with mutants (e.g., BC-box mutants that cannot assemble with the Elongin BC complex)

• Co-Immunoprecipitation of Complex Components:

  • Use ASB2 antibodies to pull down the E3 ligase complex

  • Perform western blotting for known components including Elongin B, Elongin C, Cullin5, and Rbx2

  • This approach confirms proper complex assembly and can identify new interacting partners

• Target Protein Degradation Monitoring:

  • Use ASB2 antibodies alongside antibodies against known targets (e.g., filamin B)

  • Monitor time-dependent degradation of target proteins after ASB2 induction

  • Pair with proteasome inhibitors (e.g., MG132) to confirm the proteasome-dependent nature of degradation

• Ubiquitination Site Mapping:

  • Immunoprecipitate ASB2 targets under denaturing conditions

  • Perform mass spectrometry analysis to identify ubiquitinated lysine residues

  • Compare ubiquitination patterns in the presence and absence of ASB2

• Real-Time Degradation Kinetics:

  • Use fluorescently labeled ASB2 antibodies in live-cell imaging

  • Simultaneously monitor ASB2 expression and target protein levels

  • Determine the temporal relationship between ASB2 expression and target degradation

These methodological approaches leverage ASB2 antibodies to dissect the molecular mechanisms of substrate recognition, complex formation, and ubiquitin transfer, providing insights into how ASB2 controls protein degradation during cellular differentiation processes.

What experimental approaches can reveal the role of ASB2β in muscle differentiation?

Investigating ASB2β's role in muscle differentiation requires multifaceted experimental approaches, with ASB2 antibodies being essential components of these methodologies:

• Temporal Expression Analysis During Differentiation:

  • Culture C2C12 myoblasts or primary myoblasts in differentiation medium

  • Collect samples at defined time points (0, 1, 2, 3, 5, 7 days)

  • Perform western blotting with ASB2β-specific antibodies to track expression levels

  • Correlate ASB2β expression with differentiation markers and FLNb degradation

• Loss-of-Function Studies:

  • Generate stable ASB2β knockdown in C2C12 cells using shRNAs

  • Induce differentiation and monitor:

    • Morphological changes through phase-contrast microscopy

    • Fusion index calculation (percentage of nuclei in multinucleated myotubes)

    • Expression of muscle-specific proteins (MHC, troponin T) by western blotting

    • FLNb protein levels over time

• Rescue Experiments:

  • Perform double knockdown of ASB2β and its target FLNb

  • Assess whether FLNb knockdown restores differentiation capacity in ASB2β-depleted cells

  • This approach helps establish a causal relationship between ASB2β-mediated FLNb degradation and muscle differentiation

• Structure-Function Analysis:

  • Express wild-type ASB2β and mutant variants (BC-box mutants, SOCS box mutants, UIM motif mutants)

  • Analyze their ability to restore differentiation in ASB2β-knockdown cells

  • Identify critical domains required for ASB2β function during myogenesis

• In Vivo Developmental Studies:

  • Use ASB2β antibodies for immunohistochemical analysis of developing muscle tissues

  • Track ASB2β expression during embryonic development and correlate with myogenesis stages

  • Perform conditional knockout of ASB2β in muscle precursors to assess developmental consequences

These experimental strategies, relying on high-quality ASB2β antibodies, provide comprehensive insights into how this E3 ubiquitin ligase specificity subunit regulates muscle differentiation through targeted protein degradation pathways.

How do I design experiments to study the differential targeting of filamin isoforms by ASB2α and ASB2β?

The differential targeting of filamin isoforms by ASB2α and ASB2β presents an intriguing research question that requires carefully designed experiments. Here's a methodological framework:

• Co-Immunoprecipitation Studies:

  • Express tagged versions of ASB2α or ASB2β in cellular models

  • Immunoprecipitate using ASB2 antibodies or tag-specific antibodies

  • Perform western blotting for FLNa and FLNb

  • Compare binding affinities between the different ASB2 and filamin isoforms

• Targeted Degradation Assays:

  • Express ASB2α or ASB2β in cells that express both FLNa and FLNb

  • Monitor the degradation kinetics of each filamin isoform over time

  • Include proteasome inhibitors as controls to confirm the degradation mechanism

  • Quantify degradation rates to determine preferential targeting

• Domain Mapping Experiments:

  • Generate chimeric constructs by swapping domains between ASB2α and ASB2β

  • Assess their ability to bind and degrade FLNa and FLNb

  • Focus on the unique N-terminal extension of ASB2β containing the UIM motif

  • Create point mutations in key residues to identify critical amino acids

• Ubiquitination Site Analysis:

  • Set up in vitro ubiquitination assays with purified components

  • Include ASB2α or ASB2β complexes with E1, E2 enzymes, and FLNa or FLNb substrates

  • Use mass spectrometry to identify specific lysine residues that are ubiquitinated

  • Compare ubiquitination patterns between different ASB2-filamin combinations

• Structural Analysis of ASB2-Filamin Interactions:

  • Perform pull-down assays using recombinant ASB2 domains and filamin fragments

  • Map minimal binding regions required for interaction

  • Use site-directed mutagenesis to identify key interaction interfaces

  • Consider computational docking models to predict binding modes

• Functional Consequences in Different Cell Types:

  • Examine the effects of ASB2α and ASB2β expression on:

    • Cell adhesion and spreading in hematopoietic cells vs. muscle cells

    • Cytoskeletal organization using immunofluorescence microscopy

    • Cellular differentiation markers specific to each lineage

This experimental framework allows systematic investigation of how subtle differences between ASB2 isoforms translate into differential targeting of filamin isoforms, providing insights into tissue-specific regulation of the cytoskeleton during cellular differentiation.

What controls should be included when using ASB2 antibodies in experimental workflows?

Implementing appropriate controls is crucial for obtaining reliable results when using ASB2 antibodies. Here is a comprehensive set of recommended controls for different experimental applications:

For Western Blotting:

• Positive Controls:

  • Recombinant ASB2 protein (appropriate isoform)

  • Lysates from cells known to express ASB2 (e.g., differentiating C2C12 cells for ASB2β)

  • Tagged ASB2 expressed in heterologous systems

• Negative Controls:

  • Lysates from ASB2 knockdown/knockout cells

  • Tissues/cells known not to express the specific isoform

• Antibody Controls:

  • Primary antibody omission

  • Isotype control antibody (same species and Ig class)

  • Antibody pre-absorption with immunizing peptide

For Immunoprecipitation:

• Input Sample Control: Retain a portion of the starting material

• Bead-Only Control: Perform IP procedure without primary antibody

• Irrelevant Antibody Control: Use an antibody against an unrelated protein

• Reciprocal IP: Confirm interactions by reversing the antibody used for pull-down

For Immunohistochemistry/Immunofluorescence:

• Positive Tissue Controls: Include tissues known to express ASB2

• Negative Tissue Controls: Include tissues known not to express ASB2

• Primary Antibody Omission: Process sections without primary antibody

• Blocking Peptide Control: Pre-incubate antibody with the immunizing peptide

For Functional Assays:

• Wild-type vs. Mutant Controls: Compare ASB2 wild-type with non-functional mutants (e.g., BC-box mutants)

• Rescue Experiments: Reintroduce ASB2 in knockdown cells to rescue phenotypes

• Pharmacological Controls: Use proteasome inhibitors to block degradation of ASB2 targets

For Target Validation:

• Target Overexpression: Assess effects of increasing target protein levels

• Target Knockdown: Confirm specificity by removing the proposed target

• Double Knockdown: Knock down both ASB2 and its target to assess functional relationships

These controls help distinguish specific antibody signals from background, validate protein-protein interactions, confirm functional relationships, and ultimately ensure experimental reproducibility and reliability when working with ASB2 antibodies.

How can I resolve common issues when working with ASB2 antibodies?

When working with ASB2 antibodies, researchers may encounter several technical challenges. This section provides methodological solutions to common problems:

Issue 1: Weak or Absent Signal in Western Blotting

• Methodological Solution:

  • Optimize protein extraction using buffers containing protease inhibitors to prevent ASB2 degradation

  • Enrich the sample by immunoprecipitation before western blotting

  • Increase antibody concentration incrementally (typically 1:500 to 1:100)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use a more sensitive detection system (e.g., enhanced chemiluminescence plus)

  • For isoform-specific detection, confirm you're using the appropriate antibody (e.g., 2PNAB1 serum for ASB2β)

Issue 2: Multiple Bands or Non-specific Binding

• Methodological Solution:

  • Increase blocking stringency (5% BSA or milk for 2 hours)

  • Perform more stringent washing steps (0.1% Tween-20 in TBS, 5× 10 minutes)

  • Reduce primary antibody concentration

  • Pre-absorb the antibody with cell lysates from ASB2 knockout cells

  • Verify specificity using peptide competition assays

  • Remember that ASB2 has two isoforms (α: 64 kDa, β: 70 kDa) that may both be detected

Issue 3: Inconsistent Immunoprecipitation Results

• Methodological Solution:

  • Optimize lysis conditions to maintain protein complex integrity

  • Cross-link the antibody to beads to prevent heavy chain interference

  • Use TrueBlot secondary antibodies to avoid detecting denatured IgG

  • Include detergents appropriate for membrane-associated proteins

  • Consider that ASB2 functions in a multi-protein complex - co-IP conditions should preserve these interactions

Issue 4: Poor Immunohistochemistry Staining

• Methodological Solution:

  • Optimize antigen retrieval methods (heat-induced vs. enzymatic)

  • Test different fixation protocols (4% PFA may better preserve ASB2 epitopes)

  • Use amplification systems (tyramide signal amplification)

  • Consider tissue-specific expression patterns - ASB2β is primarily in muscle tissues

  • Include positive control tissues with known ASB2 expression

Issue 5: Difficulty Detecting ASB2 During Specific Cellular States

• Methodological Solution:

  • Remember that ASB2β expression increases during myogenic differentiation - timing is critical

  • ASB2 may be rapidly degraded - use proteasome inhibitors (MG132) to stabilize

  • For inducible systems, optimize induction conditions

  • Consider the half-life of ASB2 proteins when designing time-course experiments

By systematically addressing these common issues, researchers can optimize their experimental protocols for reliable detection and analysis of ASB2 proteins across different applications.

How do I analyze ASB2 expression in relation to muscle differentiation markers?

Analyzing ASB2 expression in relation to muscle differentiation markers requires a systematic approach that correlates ASB2 levels with the progression of myogenesis. Here is a comprehensive methodological framework:

• Time-Course Analysis Protocol:

  • Culture C2C12 myoblasts in growth medium until 80-90% confluent

  • Switch to differentiation medium (typically low serum, 2% horse serum)

  • Collect samples at key timepoints: 0, 24, 48, 72, 96, and 120 hours

  • Process parallel samples for protein and RNA extraction

  • Perform western blotting with ASB2β-specific antibodies and differentiation markers

• Key Differentiation Markers to Correlate with ASB2β Expression:

  Timepoint	Early Markers	Intermediate Markers	Late Markers	ASB2β Expression
  0h (Day 0)	MyoD, Myf5 (high)	Myogenin (low)	MHC, Troponin T (absent)	Minimal
  24h (Day 1)	MyoD (high)	Myogenin (increasing)	MHC (beginning)	Increasing
  48h (Day 2)	MyoD (decreasing)	Myogenin (high)	MHC, Troponin T (present)	Moderate
  72h (Day 3)	MyoD (low)	Myogenin (high)	MHC, Troponin T (high)	High
  96h+ (Day 4+)	MyoD (low)	Myogenin (sustained)	MHC, Troponin T (very high)	High

• Correlation Analysis Methodology:

  • Quantify western blot signals using densitometry software

  • Normalize ASB2β expression to a housekeeping protein (e.g., GAPDH, β-actin)

  • Plot normalized expression levels against time

  • Calculate Pearson correlation coefficients between ASB2β and each differentiation marker

  • Identify temporal relationships (preceding, coincident, or following) between ASB2β upregulation and differentiation marker expression

• FLNb Degradation Timeline Assessment:

  • Track FLNb protein levels throughout differentiation

  • Calculate the rate of FLNb degradation in relation to ASB2β expression

  • Compare wild-type cells with ASB2β knockdown cells to confirm causality

  • Correlate FLNb degradation with myoblast fusion events

• Immunofluorescence Co-localization Studies:

  • Perform dual immunostaining for ASB2β and differentiation markers

  • Capture images at different differentiation stages

  • Analyze subcellular localization patterns

  • Quantify co-localization using digital image analysis software

• Functional Correlation through Perturbation:

  • Induce ASB2β expression at different timepoints using inducible expression systems

  • Assess the effect on differentiation marker expression

  • Determine critical windows during which ASB2β expression is necessary for proper differentiation

This comprehensive approach enables researchers to establish the precise temporal and functional relationships between ASB2β expression, target protein degradation, and the progression of muscle differentiation.

What advanced techniques can be used to study ASB2's role in ubiquitin-mediated protein degradation?

Investigating ASB2's role in ubiquitin-mediated protein degradation requires sophisticated techniques that go beyond basic biochemical assays. Here are advanced methodological approaches:

• Proximity Ligation Assay (PLA) for In Situ Ubiquitination Detection:

  • Use primary antibodies against ASB2 and ubiquitin

  • Apply species-specific PLA probes with oligonucleotide tails

  • Perform rolling circle amplification when probes are in close proximity

  • Visualize amplified signal as distinct fluorescent spots

  • This technique allows visualization of ASB2-mediated ubiquitination events in intact cells

• Tandem Ubiquitin Binding Entity (TUBE) Pull-Down:

  • Use engineered ubiquitin-binding domains with high affinity for ubiquitin chains

  • Pull down ubiquitinated proteins from cell lysates

  • Perform western blotting for ASB2 targets (e.g., FLNb)

  • Compare results between wild-type and ASB2 knockdown/knockout cells

  • This approach captures the global ubiquitination profile affected by ASB2

• CRISPR-Cas9 Genome Editing of Ubiquitination Sites:

  • Identify potential ubiquitination sites in FLNb using mass spectrometry

  • Design sgRNAs targeting these lysine residues

  • Generate knock-in mutations (K→R) that prevent ubiquitination

  • Assess protein stability and resistance to ASB2-mediated degradation

  • This approach directly tests the functional importance of specific ubiquitination sites

• Bioluminescence Resonance Energy Transfer (BRET) for Real-Time Degradation Monitoring:

  • Generate fusion proteins: target protein (e.g., FLNb) with NanoLuc luciferase and HaloTag-ASB2

  • Add NanoBRET substrate and HaloTag ligand

  • Measure energy transfer as proteins interact

  • Monitor signal decrease as the target is degraded

  • This technique allows real-time monitoring of protein-protein interactions and degradation kinetics

• Quantitative Ubiquitin Remnant Profiling:

  • Treat cells with proteasome inhibitors to accumulate ubiquitinated proteins

  • Digest proteins and enrich for peptides containing the di-glycine remnant (ubiquitination signature)

  • Perform quantitative mass spectrometry comparing cells with and without ASB2 expression

  • Identify ASB2-dependent ubiquitination sites across the proteome

  • This technique provides a global view of ASB2's impact on the ubiquitinome

• Reconstitution of ASB2 E3 Ligase Complexes In Vitro:

  • Express and purify recombinant ASB2α or ASB2β along with Elongin B, Elongin C, Cullin5, and Rbx2

  • Perform in vitro ubiquitination assays with purified components

  • Analyze reaction products using western blotting or mass spectrometry

  • Test structure-function relationships using ASB2 mutants

  • This biochemical approach allows precise control over reaction conditions and component stoichiometry

These advanced techniques provide deeper insights into the molecular mechanisms of ASB2-mediated protein degradation, allowing researchers to move beyond correlation to establish causation in regulatory pathways.

How might ASB2 antibodies be used to investigate the role of ASB2 in non-muscle tissues?

While ASB2's functions in hematopoietic and muscle cells are relatively well-characterized, its roles in other tissues remain underexplored. ASB2 antibodies are instrumental in expanding our understanding of this protein's functions across diverse tissue types:

• Comparative Tissue Expression Profiling:

  • Perform western blot analysis using pan-ASB2 and isoform-specific antibodies on protein extracts from multiple tissues

  • Create a comprehensive expression atlas of ASB2 isoforms across tissue types

  • Correlate expression patterns with tissue-specific functions

  • Unexpected expression might reveal novel roles beyond the established hematopoietic and muscle systems

• Investigation of ASB2 in Neuronal Tissues:

  • Apply immunohistochemistry with ASB2 antibodies on brain and spinal cord sections

  • Analyze co-localization with neuronal markers

  • Examine ASB2 expression during neuronal differentiation and in response to neuronal activity

  • The cytoskeletal remodeling functions of ASB2 may be relevant to neuronal plasticity and axon guidance

• ASB2 in Epithelial Tissues and Barrier Function:

  • Use immunofluorescence to examine ASB2 localization in epithelial layers

  • Correlate with markers of cell-cell junctions and adhesion complexes

  • Investigate potential roles in regulating epithelial integrity through filamin degradation

  • This approach could reveal functions in tissue barrier maintenance

• Exploration of ASB2 in Immune Cell Subsets:

  • Perform flow cytometry with fluorescently-labeled ASB2 antibodies

  • Sort immune cell populations based on ASB2 expression

  • Characterize phenotypic and functional differences between ASB2-high and ASB2-low populations

  • This may uncover roles in specific immune cell subsets beyond the known functions in myeloid differentiation

• ASB2 in Tissue Regeneration and Wound Healing:

  • Apply ASB2 antibodies to tissue sections from regeneration models

  • Track expression dynamics during healing processes

  • Correlate with markers of cellular plasticity and differentiation

  • The role of ASB2 in controlling cytoskeletal dynamics may be critical during tissue remodeling

• Investigation of ASB2 in Stem Cell Niches:

  • Use multiplexed immunofluorescence with ASB2 antibodies and stem cell markers

  • Analyze expression in quiescent versus activated stem cells

  • Examine potential roles in stem cell maintenance and differentiation potential

  • This approach could reveal functions in tissue homeostasis beyond established differentiation pathways

These research directions leverage the specificity of ASB2 antibodies to explore novel functions and regulatory mechanisms in tissues where ASB2's roles remain poorly characterized, potentially revealing new therapeutic targets for tissue-specific disorders.

What methodological approaches can reveal potential novel targets of ASB2-mediated degradation?

Identifying novel targets of ASB2-mediated degradation requires comprehensive and unbiased approaches. Here are sophisticated methodological strategies leveraging ASB2 antibodies:

• Quantitative Proteomics with Stable Isotope Labeling (SILAC):

  • Culture cells in media containing light or heavy isotope-labeled amino acids

  • Express ASB2 in one population and use control in the other

  • Mix samples, perform protein extraction, and digest into peptides

  • Analyze by mass spectrometry to identify proteins decreased upon ASB2 expression

  • Validate candidates using ASB2 antibodies for co-immunoprecipitation

• Global Protein Stability (GPS) Profiling:

  • Generate a library of GFP-tagged open reading frames

  • Express in cells with and without ASB2

  • Use fluorescence-activated cell sorting to measure protein stability

  • Identify fusion proteins degraded in an ASB2-dependent manner

  • Confirm direct interactions using reciprocal immunoprecipitation with ASB2 antibodies

• BioID Proximity Labeling:

  • Create a fusion protein of ASB2 with a biotin ligase (BirA*)

  • Express in cells and provide biotin substrate

  • BirA* will biotinylate proteins in close proximity to ASB2

  • Purify biotinylated proteins using streptavidin

  • Identify by mass spectrometry and validate using ASB2 antibodies

  • This approach captures transient interactions with potential substrates

• Ubiquitin Remnant Profiling:

  • Treat cells with proteasome inhibitors

  • Compare ASB2-expressing and control cells

  • Enrich for peptides containing ubiquitin remnant (K-ε-GG)

  • Perform quantitative mass spectrometry

  • Identify sites with increased ubiquitination in ASB2-expressing cells

  • Confirm ASB2-dependent ubiquitination using targeted assays

• CRISPR Screens for Synthetic Interactions:

  • Perform genome-wide CRISPR screens in ASB2-expressing and control cells

  • Identify genes whose loss is specifically lethal in ASB2-expressing cells

  • These may include critical ASB2 targets or pathway components

  • Validate using ASB2 antibodies to confirm physical interactions

• Yeast Two-Hybrid Screening with Domain-Specific Baits:

  • Use the ankyrin repeat domain of ASB2 as bait

  • Screen against cDNA libraries from relevant tissues

  • Identify potential binding partners

  • Validate interactions in mammalian cells using co-immunoprecipitation with ASB2 antibodies

  • Test for ASB2-dependent degradation of candidates

• Degradation Profile Comparison Between ASB2 Isoforms:

  • Express ASB2α or ASB2β in the same cellular background

  • Perform quantitative proteomics to identify isoform-specific targets

  • Use domain-swapping experiments to determine regions conferring specificity

  • This approach leverages the known differential targeting of filamin isoforms

These methodological approaches provide complementary strategies to identify the full spectrum of ASB2 substrates, potentially revealing unexpected cellular pathways regulated by ASB2-mediated protein degradation.