RFWD3 Antibody

What is RFWD3 and why is it significant in research?

RFWD3 is an E3 ubiquitin ligase that contains a RING finger domain and exhibits ubiquitin ligase activity in vitro . It plays essential roles in multiple DNA damage response pathways, particularly in translesion DNA synthesis (TLS) .

RFWD3 is significant in research because:

• It promotes ubiquitylation of proteins on single-stranded DNA (ssDNA)

• It regulates DNA damage-induced PCNA ubiquitylation

• It stimulates gap-filling DNA synthesis across different DNA lesions

• It forms complexes with critical tumor suppressors like p53 and interacts with Mdm2

• Its dysfunction has been implicated in Fanconi Anemia and potentially affects chemotherapy response in cancer

The ability to detect and study RFWD3 using antibodies is therefore crucial for understanding genome maintenance mechanisms and cellular responses to genotoxic stressors.

How should RFWD3 antibodies be validated prior to experimental use?

Validation of RFWD3 antibodies is essential before proceeding with experimental applications. Based on established practices and available literature, validation should include:

• siRNA/shRNA knockdown verification: Confirming reduced signal intensity following RFWD3 depletion, as demonstrated in multiple studies . When performing Western blot validation with RFWD3 knockdown, researchers should observe reduction in all bands detected by the antibody, as shown with RFWD3 in HGSOC cell lines .

• Molecular weight verification: RFWD3 has a calculated molecular weight of approximately 85 kDa , but multiple bands may be observed due to post-translational modifications or isoforms .

• Specificity testing across applications: If using the antibody for multiple applications (WB, IF/ICC), validation should be performed for each application independently .

• Positive and negative control tissues/cell lines: Include cell lines with known RFWD3 expression levels. For example, the COV318 cell line shows minimal RFWD3 expression compared to 59M and ES2 cell lines, making it a useful comparative control .

• Mass spectrometry confirmation: Where possible, validate specificity by confirming that only RFWD3 is significantly depleted in immunoprecipitation experiments, as demonstrated in egg extract studies .

What experimental applications are suitable for RFWD3 antibodies?

RFWD3 antibodies have been successfully employed in multiple experimental contexts:

Western Blotting (WB):

• Detecting endogenous RFWD3 expression in various cell lines (59M, ES2, COV318)

• Analyzing changes in RFWD3 levels in response to genotoxic agents or replication stress

• Identifying post-translational modifications such as phosphorylation events following DNA damage

Immunoprecipitation (IP):

• Isolating endogenous RFWD3 complexes from nuclear extracts

• Studying RFWD3 interactions with binding partners such as Mdm2 and p53

• Analyzing RFWD3-dependent protein modifications in response to DNA damage

Immunofluorescence/Immunocytochemistry (IF/ICC):

• Examining subcellular localization of RFWD3, particularly at sites of DNA damage

• Analyzing colocalization with DNA replication and repair factors

Immunodepletion:

• Removing RFWD3 from extracts to study the functional consequences on processes like translesion synthesis

Each application requires specific optimization and controls to ensure reliable results.

How can researchers optimize detection of RFWD3 phosphorylation following DNA damage?

RFWD3 undergoes phosphorylation in response to DNA damage, particularly at serine residues (S46 and S63) within SQ motifs targeted by ATR and ATM kinases . To optimize detection of these phosphorylation events:

• Timing considerations: Use appropriate timepoints when collecting samples, as ATM phosphorylates RFWD3 at early times after DNA damage, while ATR is the major kinase responsible for later phosphorylation events .

• Damage-inducing agents: Choose appropriate genotoxic agents depending on the pathway being studied:

  • Ionizing radiation (IR) for double-strand breaks and ATM activation

  • Hydroxyurea (HU) for replication stress and ATR activation

  • Camptothecin (CPT) for studying double-strand DNA breaks during replication

• Phospho-specific detection: Consider using phospho-specific antibodies when available, or employ techniques like Phos-tag gels to separate phosphorylated from non-phosphorylated forms.

• Phosphatase inhibitors: Include phosphatase inhibitors in lysis buffers to prevent dephosphorylation during sample preparation.

• Mutagenesis controls: Include phospho-site mutants (e.g., RFWD3-2SA with S46A and S63A mutations) as negative controls, as these show markedly decreased phosphorylation following IR treatment .

• Kinase overexpression: ATR overexpression can enhance RFWD3 phosphorylation, providing a positive control, while kinase-dead ATR (ATR-KD) can serve as a negative control .

What strategies can address the challenge of detecting multiple RFWD3 bands in Western blots?

Multiple RFWD3 bands in Western blots are common and likely represent different isoforms or post-translationally modified forms . To address this challenge:

• Validation through knockdown: Confirm that all bands are reduced upon RFWD3 siRNA treatment, as demonstrated in HGSOC cell lines . This helps distinguish specific bands from non-specific cross-reactivity.

• Phosphatase treatment: Treat samples with lambda phosphatase prior to SDS-PAGE to determine which bands represent phosphorylated forms.

• Size verification: Compare observed bands with the calculated molecular weight (approximately 85 kDa) and consider known modifications.

• Isoform-specific detection: When studying specific RFWD3 isoforms, design experiments to differentiate between them, potentially using isoform-specific antibodies if available.

• Sample preparation optimization: Adjust lysis conditions, buffer composition, and denaturation protocols to ensure consistent detection.

• Loading controls: Include appropriate loading controls and perform quantification across multiple biological replicates to ensure reliable comparisons between samples.

• Subcellular fractionation: Consider analyzing nuclear versus cytoplasmic fractions separately, as RFWD3 has been primarily detected in nuclear extracts .

How should researchers design experiments to study RFWD3's role in translesion DNA synthesis?

Based on the findings that RFWD3 is essential for translesion DNA synthesis across different DNA lesions , researchers should consider the following experimental design strategies:

• Model system selection: Utilize established systems such as Xenopus egg extracts, which have been successfully used to study RFWD3's function in TLS .

• Lesion-specific plasmids: Generate plasmids containing site-specific lesions like:

  • DPC (DNA-protein crosslinks) containing bacterial M.HpaII

  • Glycosylase (Fpg) covalently linked to abasic sites

  • Cyclobutane pyrimidine dimers (CPDs)

• Strand-specific analysis: Design experiments to examine both leading and lagging strand TLS by positioning lesions appropriately, as RFWD3 is essential for both .

• Nascent DNA analysis: Monitor nascent DNA extension using primer extension assays to precisely map where replication stalls in the absence of RFWD3 .

• TLS polymerase manipulation: Combine RFWD3 depletion with depletion of specific TLS polymerases (e.g., Polη or REV1-Polζ) to determine epistatic relationships .

• Immunodepletion controls: Include multiple antibodies for immunodepletion to rule out off-target effects, and confirm specificity through mass spectrometry analysis of depleted extracts .

• PCNA ubiquitylation analysis: Monitor PCNA ubiquitylation status, as RFWD3 regulates DNA damage-induced PCNA ubiquitylation, which is crucial for TLS polymerase recruitment .

What controls are essential when studying RFWD3-protein interactions using antibodies?

When investigating RFWD3 interactions with other proteins like Mdm2, p53, or replication factors, include these critical controls:

• Reciprocal immunoprecipitation: Confirm interactions by performing IP with antibodies against both RFWD3 and the putative interacting protein, as demonstrated for RFWD3-Mdm2-p53 interactions .

• Multiple antibodies: Use different antibodies recognizing distinct regions of RFWD3 (like Ab1 and Ab2 in MCF7 nuclear extracts) to validate interactions and rule out epitope-specific artifacts .

• Recombinant protein controls: Include in vitro binding assays with recombinant proteins (e.g., GST-RFWD3, His-Mdm2, Flag-p53) to confirm direct interactions .

• Domain mapping: Use deletion mutants to identify domains required for interaction, as done for the Mdm2 acidic domain required for RFWD3 binding .

• Treatment conditions: Compare interactions under normal conditions versus after genotoxic stress (IR, HU, CPT) .

• Cell line selection: Choose appropriate cell lines for studying specific interactions (e.g., p53-positive MCF7 cells versus HeLa cells with low p53) .

• Subcellular fractionation: Isolate nuclear extracts when studying nuclear interactions, as RFWD3 complexes with Mdm2 and p53 were detected in nuclear fractions .

• Non-specific binding controls: Include isotype-matched control antibodies and beads-only controls in IP experiments.

How can researchers address variability in RFWD3 expression across different cell lines?

RFWD3 expression varies significantly across cell lines, as demonstrated by the minimal expression in COV318 cells compared to 59M and ES2 cells . To address this variability:

• Baseline characterization: Thoroughly characterize RFWD3 expression in each cell line before designing experiments.

• Loading standardization: Ensure equal protein loading and include multiple housekeeping controls when comparing different cell lines.

• Multiple detection methods: Confirm expression patterns using both protein (Western blot) and mRNA (qRT-PCR) detection methods.

• Biological relevance: Consider the biological significance of expression differences, such as the correlation between low RFWD3 expression and increased chemosensitivity observed in COV318 cells .

• Antibody optimization: Optimize antibody concentration and incubation conditions for each cell line independently.

• Signal normalization: When quantifying relative expression, use appropriate normalization methods and include sufficient biological replicates.

• Genetic manipulation: Consider generating isogenic cell lines with controlled RFWD3 expression levels to eliminate confounding variables.

How should researchers interpret RFWD3 localization in relation to DNA damage sites?

RFWD3 localization at DNA damage sites provides important insights into its function. For proper interpretation:

• Correlation with replication proteins: RFWD3 recruitment strongly correlates with RPA recruitment, matching the generation of single-stranded DNA gaps during replication of damaged DNA .

• Temporal dynamics: Monitor the kinetics of RFWD3 recruitment and dissociation from damage sites, as timing can reveal functional relationships with other repair factors.

• Co-localization analysis: Perform co-localization studies with established markers:

  • RPA for ssDNA regions

  • γH2AX for double-strand breaks

  • PCNA for replication forks

• Quantitative assessment: Develop quantitative metrics for measuring RFWD3 accumulation at damage sites across different experimental conditions.

• Resolution considerations: Use appropriate microscopy techniques with sufficient resolution to distinguish between different DNA repair structures.

• Functional correlations: Correlate localization patterns with functional outcomes such as PCNA ubiquitylation or recruitment of TLS polymerases .

What methodology should be used to analyze RFWD3's role in chemotherapy response?

Based on findings linking RFWD3 to platinum chemotherapy response in cancer cells , researchers should employ these methodological approaches:

How can RFWD3 antibodies be used to study its E3 ligase activity toward different substrates?

RFWD3 functions as an E3 ubiquitin ligase that promotes ubiquitylation of proteins on ssDNA and regulates PCNA ubiquitylation . To study this activity:

• In vitro ubiquitylation assays: Use purified recombinant RFWD3 in reconstituted ubiquitylation reactions with potential substrates, detecting activity with anti-ubiquitin antibodies .

• Substrate identification: Combine RFWD3 immunoprecipitation with mass spectrometry to identify ubiquitylated proteins in RFWD3 complexes.

• PCNA ubiquitylation analysis: Monitor PCNA mono- and poly-ubiquitylation in the presence and absence of RFWD3, particularly after DNA damage .

• RPA ubiquitylation: Examine RPA ubiquitylation status, as RFWD3 has been shown to interact with RPA and potentially regulate its ubiquitylation .

• Ligase-dead controls: Include catalytically inactive RFWD3 mutants (mutations in the RING domain) as negative controls .

• Substrate specificity analysis: Compare ubiquitylation patterns of different substrates to determine RFWD3's substrate preferences and specificity.

• Chromatin association: Use chromatin fractionation combined with RFWD3 antibodies to detect substrate ubiquitylation specifically on chromatin.

What approaches can be used to study RFWD3's role in regulating replication fork remodeling?

RFWD3 promotes ZRANB3 recruitment to stalled replication forks and regulates fork remodeling . To investigate this function:

• Electron microscopy: Analyze fork structures in the presence and absence of RFWD3, looking for changes in the frequency of reversed forks .

• ZRANB3 recruitment assays: Monitor ZRANB3 localization to stalled replication forks and ubiquitinated sites of DNA damage using immunofluorescence .

• PCNA ubiquitination analysis: Examine PCNA ubiquitination status, as RFWD3 promotes PCNA ubiquitination and interaction with ZRANB3 .

• Epistasis analysis: Compare the effects of RFWD3 and ZRANB3 depletion individually and in combination to establish their functional relationship .

• Fork protection assays: Assess nascent DNA degradation in BRCA2-deficient cells with and without RFWD3, as RFWD3 inactivation rescues fork degradation similar to ZRANB3 inactivation .

• USP1 inhibition studies: Analyze ZRANB3 foci formation after inhibition of the PCNA deubiquitinase USP1 in the presence and absence of RFWD3 .

• Interaction domain mapping: Identify the domains of RFWD3 required for promoting ZRANB3 recruitment to better understand the mechanism.

Table 1: Correlation between RFWD3 expression and carboplatin sensitivity in ovarian cancer cell lines. Data derived from expression and chemosensitivity analysis .