RFWD2 Antibody

What is RFWD2 and what are its primary cellular functions?

RFWD2 (also known as COP1) is an E3 ubiquitin ligase that plays crucial roles in protein degradation pathways. It regulates multiple cellular processes including cell cycle progression, cell growth, and apoptosis. RFWD2 has been identified as an important regulator in the ubiquitin-proteasome system, where it targets specific proteins for degradation. In multiple myeloma, RFWD2 has been shown to control cellular proliferation via regulating the degradation of P27 rather than P53, and it mediates P27 ubiquitination by interacting with RCHY1 . Additionally, RFWD2 plays a significant role in developmental processes, particularly in lung branching morphogenesis through protein-level regulation of transcription factors .

What are the key specifications of commercially available RFWD2 antibodies?

Commercial RFWD2 antibodies are typically available as rabbit polyclonal antibodies that recognize human, mouse, and rat RFWD2. For example, the DF4023 RFWD2 antibody is a rabbit polyclonal with applications in Western blot (WB), immunohistochemistry (IHC), and immunofluorescence/immunocytochemistry (IF/ICC). The molecular weight of the target protein is approximately 80 kDa. When selecting an antibody, researchers should consider the specific applications needed (WB, IHC, IF/ICC), species reactivity (human, mouse, rat), and potential cross-reactivity with predicted species such as bovine and chicken .

What are the optimal conditions for using RFWD2 antibody in Western blotting?

For optimal Western blot detection of RFWD2:

• Sample preparation: Use standard protein extraction procedures with protease inhibitors to prevent protein degradation.

• Gel electrophoresis: Run samples on 8-10% SDS-PAGE gels to properly resolve the 80 kDa RFWD2 protein.

• Transfer: Use PVDF membranes for optimal protein binding.

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute RFWD2 antibody according to manufacturer recommendations (optimal dilutions should be determined by the end user). Typical starting dilutions range from 1:500 to 1:2000.

• Detection: HRP-conjugated secondary antibodies with enhanced chemiluminescence (ECL) detection systems are recommended.

• Expected result: A band at approximately 80 kDa corresponding to RFWD2 .

When analyzing RFWD2 in cancer samples, particularly multiple myeloma, include appropriate positive and negative controls to establish baseline expression levels .

What approaches should be used for RFWD2 antibody validation in research applications?

A comprehensive validation approach for RFWD2 antibody should include:

• Positive and negative tissue/cell controls: Based on known expression patterns (higher in testis, placenta, skeletal muscle, and heart).

• Overexpression controls: Using RFWD2-overexpressing cell lines (as described in multiple myeloma studies with ARP1 and H929 cells) .

• Knockdown controls: Using RFWD2-shRNA transfected cells to confirm antibody specificity.

• Western blot analysis: Confirming single band of expected size (80 kDa).

• Peptide competition assay: Pre-incubating antibody with immunizing peptide to confirm specificity.

• Cross-reactivity assessment: Testing against related proteins to ensure specificity.

• Reproducibility testing: Consistent results across multiple experiments and lots .

How should RFWD2 antibodies be optimized for immunohistochemistry in tissue samples?

For optimal immunohistochemical detection of RFWD2:

• Fixation: 10% neutral buffered formalin is generally suitable, but optimization may be required for specific tissues.

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). Test both to determine optimal conditions.

• Blocking: Use appropriate blocking solution to reduce non-specific binding (3% BSA or serum from the same species as the secondary antibody).

• Primary antibody incubation: Begin with manufacturer's recommended dilution (typically 1:100 to 1:500) and optimize as needed. Incubate overnight at 4°C.

• Detection system: Use a sensitive detection system appropriate for your tissue (e.g., polymer-based detection systems).

• Counterstaining: Hematoxylin for nuclear visualization.

• Controls: Include known positive tissues (testis, placenta) and negative controls (primary antibody omission) .

For lung tissue studies, particularly in developmental contexts, special attention should be paid to fixation time and antigen retrieval to preserve tissue architecture while allowing for adequate antibody access .

How is RFWD2 implicated in multiple myeloma progression and what methodologies are crucial for its study?

RFWD2 plays a significant role in multiple myeloma (MM) progression:

What are the recommended experimental designs for studying RFWD2's role in drug resistance mechanisms?

To effectively study RFWD2's role in drug resistance:

• Cell line selection: Use established drug-sensitive and resistant myeloma cell lines (e.g., ARP1, H929) for comparative studies.

• Modulation of RFWD2 expression:

  • Overexpression: Transfect cells with CRISPR lentiviral activation particles

  • Knockdown: Use lentiviral shRNA transfection technology

• Drug sensitivity testing:

  • Treat cells with proteasome inhibitors (e.g., bortezomib)

  • Measure cell viability, proliferation, and apoptosis

  • Determine IC50 values before and after RFWD2 modulation

• Mechanistic studies:

  • Analyze P27 protein levels and ubiquitination status

  • Investigate RFWD2-RCHY1 interaction

  • Examine cell cycle progression using flow cytometry

• In vivo validation:

  • Generate xenograft mouse models using BTZ-resistant MM cells

  • Test the effect of RFWD2 inhibition on tumor growth and drug response

• Clinical correlation:

  • Compare RFWD2 expression between paired baseline/relapse patient samples

  • Correlate expression with clinical outcomes and treatment responses

How does RFWD2 expression pattern differ between newly diagnosed and relapsed multiple myeloma patients?

Analysis of 88 paired baseline/relapse samples has demonstrated a significant difference in RFWD2 expression:

What techniques are recommended for studying RFWD2's role in lung development and branching morphogenesis?

To investigate RFWD2's function in lung development:

• Expression analysis:

  • Immunohistochemistry with RFWD2 antibody on developing lung sections

  • RT-qPCR for temporal expression patterns during development

  • In situ hybridization to visualize spatial expression patterns

• Genetic manipulation strategies:

  • Conditional knockout models to study tissue-specific effects

  • Time-specific inactivation to determine critical developmental windows

• Branching morphogenesis analysis:

  • Ex vivo lung explant cultures to visualize branching in real-time

  • Whole-mount immunostaining with epithelial markers (Sox2 for proximal, Sox9 for distal epithelium)

  • 3D reconstruction and quantitative analysis of branching patterns

• Molecular pathway analysis:

  • Co-immunoprecipitation to identify RFWD2 interaction partners

  • Protein degradation assays to determine ubiquitination targets

  • Transcription factor activity assays (particularly for ETV transcription factors)

• Functional assessment:

  • Respiratory function tests in animal models

  • Histological analysis of lung architecture

  • Lineage tracing to determine cell fate decisions

How can researchers effectively investigate the interaction between RFWD2 and its target proteins?

To study RFWD2's interactions with target proteins:

• Co-immunoprecipitation (Co-IP):

  • Use anti-RFWD2 antibody to pull down RFWD2 and associated proteins

  • Confirm interactions by Western blot with antibodies against suspected targets

  • For multiple myeloma research, focus on P27 and RCHY1 interactions

• Proximity ligation assay (PLA):

  • Visualize protein interactions in situ

  • Use RFWD2 antibody in combination with antibodies against potential targets

  • Quantify interaction signals in different cellular compartments

• Mass spectrometry-based approaches:

  • Immunoprecipitate RFWD2 and identify interacting proteins

  • Quantitative proteomics to assess changes in protein abundance following RFWD2 modulation

  • Analysis of ubiquitinated proteome to identify RFWD2 substrates

• In vitro ubiquitination assays:

  • Reconstitute ubiquitination reactions with purified components

  • Detect ubiquitinated products by Western blot

  • Identify ubiquitination sites by mass spectrometry

• FRET/BRET assays:

  • Generate fluorescent fusion proteins to visualize interactions in living cells

  • Measure protein-protein interactions in real-time

  • Determine subcellular localization of interactions

What are the most common technical challenges when working with RFWD2 antibodies and how can they be addressed?

Common challenges and solutions:

• High background in Western blot:

  • Increase blocking time and concentration (5-10% blocking solution)

  • Optimize antibody dilutions (perform titration experiments)

  • Increase wash duration and number of wash steps

  • Use different blocking agents (milk vs. BSA)

  • Consider using more sensitive detection systems

• Multiple bands in Western blot:

  • Verify sample preparation (include protease inhibitors)

  • Test antibody specificity using knockdown controls

  • Optimize SDS-PAGE conditions

  • Consider post-translational modifications or isoforms of RFWD2

• Weak or no signal in IHC:

  • Optimize antigen retrieval methods (test different buffers and conditions)

  • Increase antibody concentration or incubation time

  • Use amplification systems (e.g., tyramide signal amplification)

  • Verify tissue fixation protocols

  • Confirm RFWD2 expression in the tissue of interest

• Inconsistent results across experiments:

  • Standardize protocols and reagents

  • Use positive controls in each experiment

  • Consider lot-to-lot variability in antibodies

  • Monitor storage conditions and avoid freeze-thaw cycles

How can researchers design effective experiments to differentiate between RFWD2's role as an oncogene versus tumor suppressor?

RFWD2 has shown both oncogenic and tumor suppressive functions in different cancer contexts. To differentiate these roles:

• Context-specific expression analysis:

  • Compare RFWD2 expression across multiple cancer types

  • Correlate expression with clinical outcomes in each cancer type

  • Studies show tumor suppressor roles in prostate and gastric cancers versus oncogenic roles in hepatocellular carcinoma, breast cancer, ovarian adenocarcinoma, and acute myeloid leukemia

• Functional assays with bidirectional modulation:

  • Perform both overexpression and knockdown experiments in the same model

  • Measure effects on proliferation, apoptosis, migration, and invasion

  • Assess drug sensitivity changes following RFWD2 modulation

• Target specificity analysis:

  • Investigate effects on both tumor suppressors (e.g., p53) and oncogenes (e.g., JUN)

  • Use ChIP-seq to identify genomic binding sites

  • Perform RNA-seq after RFWD2 modulation to identify transcriptional changes

• In vivo models:

  • Generate tissue-specific transgenic and knockout models

  • Compare tumor initiation, progression, and metastasis

  • Assess therapeutic responses in different genetic backgrounds

• Mechanistic investigation:

  • Identify tissue-specific interaction partners

  • Determine ubiquitination substrates in different contexts

  • Analyze pathway activation using phospho-specific antibodies

What are the best approaches for studying post-translational modifications of RFWD2 and their impact on its function?

To investigate post-translational modifications (PTMs) of RFWD2:

• Identification of PTMs:

  • Immunoprecipitate RFWD2 using specific antibodies

  • Analyze by mass spectrometry to identify phosphorylation, ubiquitination, SUMOylation, or other modifications

  • Use phospho-specific or modification-specific antibodies if available

• Functional significance:

  • Generate mutants at modification sites (e.g., phospho-mimetic or phospho-deficient)

  • Test effects on RFWD2 activity, stability, localization, and protein interactions

  • Identify upstream regulators (kinases, phosphatases, or other modifying enzymes)

• Spatiotemporal regulation:

  • Use immunofluorescence with modification-specific antibodies

  • Analyze PTM patterns during cell cycle or in response to cellular stresses

  • Examine modifications in different subcellular compartments

• Impact on target specificity:

  • Determine how PTMs affect RFWD2's ability to recognize and ubiquitinate substrates

  • Perform in vitro ubiquitination assays with modified and unmodified RFWD2

  • Analyze substrate binding using co-IP or surface plasmon resonance

• Therapeutic implications:

  • Identify drugs that modulate RFWD2 PTMs

  • Test whether PTM status affects response to proteasome inhibitors

  • Develop strategies to target specific modified forms of RFWD2

What new methodologies are being developed to target RFWD2 for therapeutic purposes in cancer?

Emerging approaches for targeting RFWD2 therapeutically:

• Small molecule inhibitors:

  • Design compounds that disrupt RFWD2's E3 ligase activity

  • Target specific protein-protein interactions (e.g., RFWD2-RCHY1 interaction)

  • Develop allosteric modulators that alter RFWD2 conformation

• Proteolysis-targeting chimeras (PROTACs):

  • Design bifunctional molecules that bind RFWD2 and recruit other E3 ligases

  • Induce RFWD2 degradation through the ubiquitin-proteasome system

  • Achieve tissue-specific targeting through appropriate warheads

• Gene therapy approaches:

  • Develop CRISPR/Cas9 strategies to edit RFWD2 expression

  • Use RNA interference to temporarily downregulate RFWD2

  • Employ antisense oligonucleotides to modulate RFWD2 splicing

• Combination therapies:

  • In multiple myeloma, combine RFWD2 inhibition with proteasome inhibitors

  • Target RFWD2 together with its downstream effectors

  • Research indicates that blocking RFWD2 in BTZ-resistant MM cells can overcome drug resistance in xenograft mouse models

• Biomarker-guided approaches:

  • Stratify patients based on RFWD2 expression levels

  • Tailor treatment strategies based on RFWD2 status

  • Monitor RFWD2 expression during treatment to detect resistance development

How can multi-omics approaches enhance our understanding of RFWD2 function in different biological contexts?

Integrated multi-omics strategies for RFWD2 research:

• Genomics and transcriptomics integration:

  • Correlate RFWD2 genetic alterations with expression changes

  • Identify transcriptional networks regulated by RFWD2

  • Analyze alternative splicing patterns of RFWD2 across tissues

• Proteomics and ubiquitinomics:

  • Global proteome analysis after RFWD2 modulation

  • Ubiquitin remnant profiling to identify direct ubiquitination targets

  • Quantitative analysis of protein turnover rates

• Metabolomics integration:

  • Assess metabolic changes associated with RFWD2 function

  • Identify metabolic pathways affected by RFWD2-mediated protein degradation

  • Connect metabolic alterations to cellular phenotypes

• Spatial transcriptomics and proteomics:

  • Map RFWD2 expression and function in tissue microenvironments

  • Analyze cell-type specific roles in complex tissues

  • Particularly valuable for developmental biology studies (e.g., lung branching)

• Single-cell multi-omics:

  • Characterize heterogeneity in RFWD2 function at single-cell resolution

  • Identify cell populations particularly dependent on RFWD2

  • Track dynamic changes during development or disease progression

• Network biology approaches:

  • Construct integrated networks incorporating multiple data types

  • Identify central nodes and pathways regulated by RFWD2

  • Predict functional consequences of RFWD2 perturbation