SH3RF1 Antibody, HRP conjugated

What is SH3RF1 and what cellular functions does it regulate?

SH3RF1, also known as POSH (Plenty of SH3 domains), is an E3 ubiquitin-protein ligase containing multiple protein-protein interaction SH3 domains and a RING finger domain that confers E3 ligase activity. SH3RF1 acts as a negative post-translational regulator of proteins like FAT1, controlling their expression levels and stabilization at the cell surface. Studies have shown that siRNA-mediated knockdown of SH3RF1 increases FAT1 protein levels and stabilizes its expression at the cell surface, while overexpression of SH3RF1 reduces FAT1 levels . SH3RF1 belongs to the SH3RF/POSH family, which also includes SH3RF2/POSHER and SH3RF3, all sharing similar domain structures including SH3 domains and RING domains that provide E3 ligase activity . These proteins play important roles in cellular signaling pathways, particularly in regulatory processes related to protein ubiquitination, cell growth, and cell division.

What experimental approaches are most effective for studying SH3RF1 interactions?

For studying SH3RF1 protein interactions, researchers should consider multiple complementary approaches to validate findings. Yeast two-hybrid screening has proven effective, as demonstrated in the identification of SH3RF1 as a binding partner of the FAT1 cytoplasmic tail . For mammalian systems, co-immunoprecipitation followed by Western blotting provides direct evidence of protein-protein interactions. When designing co-IP experiments, researchers should:

• Use stringent washing conditions to minimize non-specific binding

• Include appropriate negative controls (IgG, isotype controls)

• Consider both N- and C-terminal tagged constructs, as SH3RF1's functional domains may be masked by tags

• Validate interactions using reciprocal co-IPs where possible

DHFR-PCA (dihydrofolate reductase protein-fragment complementation assay) can also be employed to detect SH3 domain-mediated interactions in live cells, though researchers should be aware that domain deletions or swapping can alter protein folding or positioning within complexes, potentially affecting binding preferences .

How do SH3RF1 antibodies compare in specificity to antibodies against other SH3 domain-containing proteins?

SH3 domains are highly conserved protein-protein interaction modules found in many proteins, which creates challenges for antibody specificity. When selecting SH3RF1 antibodies, researchers should carefully evaluate:

• The immunogen used to generate the antibody (peptide vs. recombinant protein)

• Validation data against multiple cell lines/tissues

• Cross-reactivity testing with other SH3 domain-containing proteins

Antibodies raised against unique regions outside the SH3 domains typically offer better specificity. For example, antibodies targeting the specific amino acid sequence between SH3 domains or the RING domain of SH3RF1 may provide better discrimination from related proteins like SH3RF2/POSHER and SH3RF3. Comparatively, antibodies targeting the highly conserved SH3 domains themselves may exhibit cross-reactivity with other family members (SH3GL1/Endophilin A2) and should be validated thoroughly before use in critical experiments .

What are the optimal conditions for Western blot applications using HRP-conjugated SH3RF1 antibodies?

For optimal Western blotting with HRP-conjugated SH3RF1 antibodies, consider the following protocol adjustments:

For the SH3RF1 protein specifically, include positive controls such as lysates from cells with known SH3RF1 expression and negative controls using lysates from SH3RF1 knockdown cells or tissues. Since SH3RF1 functions as an E3 ligase, be aware that multiple bands may represent different ubiquitination states or isoforms of the protein.

What fixation and permeabilization protocols are recommended for immunofluorescence detection of SH3RF1?

Based on validated immunofluorescence protocols, the following conditions are recommended for optimal SH3RF1 detection:

• Fixation: 4% formaldehyde in PBS for 15 minutes at room temperature

• Permeabilization: 0.2% Triton X-100 in PBS for 10 minutes

• Blocking: 10% normal goat serum in PBS for 1 hour

• Primary antibody incubation: Anti-SH3RF1 antibody at 1:200-1:500 dilution overnight at 4°C

• Secondary antibody: Fluorophore-conjugated secondary antibody (if using non-HRP conjugated primary) or direct visualization (if using HRP-conjugated antibody with tyramide signal amplification)

This protocol has been validated in HeLa cells for SH3RF1 antibody (PACO59197) . When using HRP-conjugated antibodies for immunofluorescence, consider implementing tyramide signal amplification (TSA) for enhanced sensitivity, especially for detecting low-abundance targets.

How can researchers optimize blocking conditions to minimize background with HRP-conjugated antibodies?

Background signal is a common challenge when using HRP-conjugated antibodies. To minimize background while maintaining specific signal:

• Test multiple blocking agents:

  • 3-5% BSA in TBS/PBS for lower background in some applications

  • 5% non-fat dry milk in TBS/PBS for general blocking

  • Commercial blocking buffers specifically formulated for HRP detection systems

• Add 0.1-0.3% Tween-20 to washing and antibody diluent buffers to reduce non-specific hydrophobic interactions

• For tissues or cells with high endogenous peroxidase activity, pre-treat with hydrogen peroxide (3% H₂O₂ for 10 minutes) before applying the primary antibody

• Consider including mild protein denaturants (0.1-0.3% SDS) in blocking solutions when dealing with high background, though this may affect some epitopes

• For tissues with high biotin content, include an avidin/biotin blocking step if using biotinylated detection systems

Empirical testing of these conditions with proper controls is essential for optimizing signal-to-noise ratio in each experimental system.

How can SH3RF1 antibodies be used to investigate its role in protein regulation?

SH3RF1 functions as a negative regulator of protein levels through its E3 ubiquitin ligase activity. To investigate this regulatory role:

• Protein stability assays: Treat cells with cycloheximide to inhibit protein synthesis, then harvest at different time points to assess degradation rates of potential SH3RF1 targets with and without SH3RF1 knockdown/overexpression. Western blotting with SH3RF1 antibodies can confirm knockdown/overexpression efficiency.

• Ubiquitination assays: Immunoprecipitate potential target proteins (e.g., FAT1) followed by Western blotting with anti-ubiquitin antibodies to detect ubiquitination under conditions of SH3RF1 modulation.

• Cell surface protein analysis: Use biotinylation of surface proteins followed by pull-down with Neutravidin and Western blot analysis to quantify cell surface expression of target proteins. This approach has demonstrated that SH3RF1 knockdown increases FAT1 levels at the cell surface .

• Co-localization studies: Perform dual immunofluorescence with SH3RF1 antibodies and antibodies against potential target proteins to visualize their spatial relationship in cells, particularly in cellular compartments associated with protein degradation.

By combining these approaches, researchers can establish both physical interactions and functional consequences of SH3RF1-mediated regulation on target proteins.

What considerations are important when designing SH3RF1 knockdown validation experiments?

When validating SH3RF1 knockdown efficiency for functional studies:

• Multiple siRNA sequences: Use at least 2-3 different siRNA sequences targeting SH3RF1 to control for off-target effects. Consistent phenotypes across different siRNAs strengthen confidence in the specificity of the observed effects.

• Quantification methods: Validate knockdown at both mRNA level (qPCR) and protein level (Western blot with SH3RF1 antibodies). This dual validation is particularly important for proteins with long half-lives.

• Time course analysis: Monitor knockdown efficiency over time (24h, 48h, 72h post-transfection) to determine the optimal window for functional assays.

• Rescue experiments: Perform rescue experiments with siRNA-resistant SH3RF1 constructs to confirm that observed phenotypes are specifically due to SH3RF1 depletion.

• Functional readouts: For SH3RF1 specifically, measure levels of known targets like FAT1 as functional validation of effective knockdown. Increased FAT1 levels at the cell surface serve as a positive control for SH3RF1 functional depletion .

These approaches help ensure that observed phenotypes are specifically attributable to SH3RF1 depletion rather than off-target effects or insufficient knockdown.

What are common causes of inconsistent results when using SH3RF1 antibodies?

Inconsistent results with SH3RF1 antibodies can stem from several factors:

• Epitope masking: SH3RF1's conformation or interactions with other proteins may mask antibody epitopes in certain contexts. Try multiple antibodies targeting different regions of SH3RF1.

• Post-translational modifications: SH3RF1 may undergo auto-ubiquitination or other modifications that affect antibody recognition. Use phosphatase or deubiquitinase treatments in parallel samples to assess this possibility.

• Expression levels: Endogenous SH3RF1 expression varies across cell types and conditions. Preliminary experiments should establish detection thresholds in your specific model system.

• Sample preparation variables: Inconsistent lysis conditions can affect protein extraction efficiency. Standardize lysis buffers, incubation times, and protein quantification methods.

• Antibody batch variation: Different lots of antibodies may have varying affinities or specificities. Document lot numbers and test new lots against old lots when possible.

• SH3 domain cross-reactivity: The SH3 domains in SH3RF1 share homology with other proteins like SH3GL1/Endophilin A2 . Confirm antibody specificity using knockout/knockdown controls.

Implementing rigorous controls and standardized protocols can help minimize these sources of variability.

How should researchers interpret data from SH3RF1 domain-swapping experiments?

Domain-swapping experiments provide valuable insights into SH3RF1 function, but data interpretation requires careful consideration:

• Context-dependency: The ability of an SH3 domain to mediate protein-protein interactions is highly dependent on its host protein context and its position within that protein. Studies have shown that swapping SH3 domains between proteins can lead to unexpected gains or losses of interactions .

• Protein folding effects: Domain swapping may alter protein folding or positioning within complexes, resulting in gained interactions that were not detected with the native protein. These should not be dismissed as artifacts but considered as potential interactions that may occur under specific conditions in vivo .

• Quantitative analysis: When analyzing domain-swapping experiments, consider both the number of interactions lost and gained. A high correlation between swap mutant and wild-type interaction profiles suggests the domain contributes minimally to specificity in that context.

• Functional validation: Complement interaction data with functional assays to determine whether gained/lost interactions have biological consequences. For example, hygromycin resistance assays have been used to validate the functional significance of SH3 domain-dependent interactions .

• Evolutionary context: Consider the evolutionary relationship between swapped domains. Domains with higher sequence similarity are more likely to functionally substitute for each other, as demonstrated by clustering analysis of SH3 domain swap effects .

By integrating these considerations, researchers can extract maximum biological insight from domain-swapping experiments with SH3RF1.

How can researchers investigate the role of SH3RF1 in disease contexts?

Given the potential involvement of SH3RF family members in diseases like Alzheimer's, several approaches can be applied to study SH3RF1 in disease contexts:

• Genetic association studies: Analyze whether SH3RF1 variants correlate with disease risk or progression, similar to how SH3RF3 variants have been associated with delayed onset of Alzheimer's disease in PSEN1 mutation carriers .

• Pathway analysis: Investigate SH3RF1's role in signaling pathways implicated in disease, particularly JNK and NFκB pathways. Approaches include:

  • Phospho-specific antibodies to detect JNK/NFκB activation states

  • Reporter assays to measure pathway activity

  • Co-immunoprecipitation to identify disease-specific interaction partners

• Cell-type specific functions: Given that SH3RF3 shows different effects in neurons versus microglia in Alzheimer's disease models , investigate SH3RF1 expression and function across different cell types in disease-relevant tissues.

• Inflammatory responses: Since SH3RF3 knockdown reduces inflammatory cytokine production in microglia responding to Aβ42 oligomers , examine whether SH3RF1 similarly modulates inflammatory responses in relevant disease models.

• Therapeutic targeting assessment: Evaluate whether modulating SH3RF1 levels or activity affects disease-related phenotypes, using approaches like RNAi, CRISPR/Cas9 editing, or small molecule inhibitors targeting SH3RF1's E3 ligase activity.

These approaches can help establish whether SH3RF1 plays causal roles in disease processes or represents a potential therapeutic target.

What methodologies are suitable for studying SH3RF1's E3 ligase activity?

To investigate SH3RF1's E3 ubiquitin ligase function:

• In vitro ubiquitination assays: Reconstitute the ubiquitination reaction using:

  • Purified recombinant SH3RF1 (full-length or RING domain)

  • E1 and E2 enzymes

  • Ubiquitin (wild-type or mutant forms)

  • ATP regeneration system

  • Potential substrate proteins

  Monitor ubiquitination by Western blotting with anti-ubiquitin antibodies or using labeled ubiquitin.

• Cell-based ubiquitination assays:

  • Express tagged ubiquitin and potential substrates

  • Immunoprecipitate substrate under denaturing conditions

  • Detect ubiquitinated species by Western blotting

• RING domain mutant controls: Generate RING domain mutants (e.g., by mutating critical zinc-coordinating residues) to serve as catalytically inactive controls.

• Ubiquitin chain topology analysis: Use ubiquitin mutants (K48R, K63R, etc.) or linkage-specific antibodies to determine the type of ubiquitin chains generated by SH3RF1, which can provide insight into the functional outcome (degradation vs. signaling).

• Proteasome inhibition experiments: Treat cells with proteasome inhibitors (e.g., MG132) to determine if SH3RF1-mediated ubiquitination targets proteins for proteasomal degradation.

These approaches can establish both the enzymatic activity of SH3RF1 and the functional consequences of this activity on specific substrate proteins.

How can researchers distinguish between direct and indirect effects in SH3RF1 functional studies?

Differentiating direct from indirect effects is particularly challenging when studying multidomain scaffolding proteins like SH3RF1. Recommended approaches include:

• Domain-specific mutants: Generate mutants affecting specific functions of SH3RF1:

  • RING domain mutants to disable E3 ligase activity

  • SH3 domain point mutations that disrupt specific protein-protein interactions

  • Combined mutations to assess domain interdependencies

• Temporal resolution: Use rapid induction systems (e.g., auxin-inducible degron) to achieve acute depletion of SH3RF1, minimizing compensatory responses and secondary effects.

• In vitro reconstitution: Demonstrate direct effects using purified components in cell-free systems, particularly for enzymatic activities and direct binding interactions.

• Proximity labeling: Employ BioID or APEX2 fused to SH3RF1 to identify proteins in close proximity in living cells, helping to distinguish direct binding partners from downstream effectors.

• Quantitative phosphoproteomics: Compare phosphorylation changes following SH3RF1 depletion with and without inhibitors of key signaling pathways (e.g., JNK, NFκB) to map direct vs. indirect signaling consequences.

By systematically applying these approaches, researchers can build a hierarchical model of SH3RF1's direct interactors and downstream effectors.

What emerging technologies might advance SH3RF1 research in the near future?

Several cutting-edge technologies hold promise for advancing SH3RF1 research:

• Cryo-EM and structural biology: Determining the structure of full-length SH3RF1 alone and in complex with binding partners would provide critical insights into conformational regulation and binding specificity.

• Single-cell multi-omics: Combining transcriptomics, proteomics, and phosphoproteomics at single-cell resolution could reveal cell-type specific roles of SH3RF1 in heterogeneous tissues.

• Live-cell imaging of ubiquitination: New biosensors for monitoring protein ubiquitination in living cells could enable real-time visualization of SH3RF1 E3 ligase activity.

• Induced proximity approaches: Technologies like PROTAC or molecular glues could be developed to target SH3RF1 for selective degradation or to modulate its interactions with specific partners.

• Cell-type specific proteomics: Techniques like PRO-CLIP (proximity-dependent cell-type-specific proteomics) could identify SH3RF1 interaction partners in specific cell types within complex tissues.

• AlphaFold-based modeling: Computational prediction of SH3RF1 structure and interaction interfaces could guide rational design of inhibitors or interactors targeting specific functions.

These technologies, alone or in combination, will likely provide new mechanistic insights into SH3RF1 function and potentially reveal novel therapeutic approaches for targeting its activity in disease contexts.