RNF125 Antibody, FITC conjugated

What is RNF125 and what are its key biological functions?

RNF125 (Ring Finger Protein 125) functions as an E3 ubiquitin-protein ligase that plays crucial roles in multiple cellular processes. It acts as a positive regulator of T-cell activation and mediates ubiquitination and subsequent proteasomal degradation of target proteins . Recent studies have identified RNF125 as a negative regulator of PD-L1 expression, where it promotes K48-linked polyubiquitination of PD-L1 and mediates its degradation . Additionally, RNF125 is implicated in innate immune responses to viral infections through regulation of DDX58/RIG-I and has been shown to interact with and regulate JAK1 stability . The protein has gained significant attention in cancer research, particularly in melanoma where its downregulation is associated with resistance to BRAF inhibitors .

What specific region does the FITC-conjugated RNF125 antibody recognize and why is this important?

The FITC-conjugated RNF125 antibody (ABIN7151132) specifically recognizes amino acids 143-231 of the human RNF125 protein . This region is particularly significant as it contains functional domains critical for RNF125's biological activity. When designing experiments, understanding the epitope location helps researchers interpret results, especially when studying protein interactions or functional domains. The antibody's specificity for this region enables precise detection of RNF125 in experimental settings where visualization of the protein is needed while maintaining its natural interactions with binding partners .

What are the recommended storage and handling conditions for FITC-conjugated RNF125 antibodies?

FITC-conjugated RNF125 antibodies should be stored at -20°C or -80°C to maintain optimal activity . Repeated freeze-thaw cycles should be avoided as they can compromise antibody integrity and fluorophore stability. The antibody is supplied in a liquid format with a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . When working with this antibody, researchers should note that it contains ProClin, which is classified as a hazardous substance requiring appropriate handling precautions by trained personnel . For long-term storage, aliquoting the antibody before freezing is recommended to minimize freeze-thaw cycles and preserve the FITC fluorescence intensity.

What experimental approaches are optimal for studying RNF125's ubiquitination activity using fluorescence-based detection?

For studying RNF125's ubiquitination activity using FITC-conjugated antibodies, researchers should implement a multi-faceted approach:

• Co-Immunoprecipitation with Fluorescence Detection: Perform Co-IP experiments similar to those described in the literature, where endogenous RNF125 was detected in protein complexes with potential substrates like PD-L1 . The FITC-conjugated antibody can be used for direct visualization of RNF125 in the immunoprecipitated complexes using fluorescence-based detection methods.

• Ubiquitination Assays: Set up in vitro or cell-based ubiquitination assays with potential RNF125 substrates (e.g., PD-L1, JAK1) co-expressed with tagged ubiquitin . After immunoprecipitation of the substrate, the FITC-conjugated RNF125 antibody can be used to confirm the presence of RNF125 in the ubiquitination complex.

• Confocal Microscopy: Utilize the FITC-conjugated antibody for confocal microscopy to visualize the co-localization of RNF125 with its substrates before and after stimulation of ubiquitination pathways. This approach was successfully used to show that PD-L1 staining intensity decreased following RNF125 overexpression .

• FRET Analysis: For advanced applications, researchers can pair the FITC-conjugated RNF125 antibody with a compatible acceptor fluorophore-labeled substrate antibody to perform Förster Resonance Energy Transfer (FRET) analysis, providing real-time visualization of protein-protein interactions in living cells.

How can researchers effectively use this antibody to investigate RNF125's role in cancer immunotherapy resistance?

To investigate RNF125's role in cancer immunotherapy resistance using FITC-conjugated antibodies, researchers should consider these methodological approaches:

• Flow Cytometry Analysis: Use the FITC-conjugated RNF125 antibody to quantify RNF125 expression levels in patient-derived tumor samples or cell lines with varying responses to immunotherapy. This approach enables correlation of RNF125 expression with clinical outcomes or resistance patterns .

• Fluorescence Microscopy in Tumor Sections: Apply the antibody in immunofluorescence studies of tumor sections to examine the spatial relationship between RNF125 expression and immune cell infiltration. This is particularly relevant given the observed correlation between RNF125 expression and CD4+, CD8+ T cell and macrophage tumor infiltration .

• Live-Cell Imaging: For dynamic studies, use the FITC-conjugated antibody in live-cell imaging experiments to track RNF125 localization and expression changes in response to immunotherapy treatments.

• Dual Staining Protocols: Implement dual staining protocols that combine the FITC-conjugated RNF125 antibody with antibodies against PD-L1 or other immune checkpoint molecules to assess their inverse relationship in tumor samples, as demonstrated in previous research showing RNF125 negatively regulates PD-L1 levels .

• RNF125 Knockout/Overexpression Models: Generate RNF125 knockout or overexpression models in tumor cell lines, then use the FITC-conjugated antibody to confirm altered expression before assessing changes in immunotherapy response, similar to the approach where RNF125 knockout in MC-38 and H22 cells led to higher PD-L1 levels and faster tumor growth .

What are the critical controls needed when using FITC-conjugated RNF125 antibodies in immunofluorescence studies?

When conducting immunofluorescence studies with FITC-conjugated RNF125 antibodies, the following critical controls should be implemented:

These controls ensure reliable, reproducible results and proper interpretation of RNF125 localization and expression data, particularly important given the complex regulatory roles of RNF125 in immune and cancer contexts .

How can researchers optimize flow cytometry protocols when working with FITC-conjugated RNF125 antibodies for intracellular staining?

Optimizing flow cytometry protocols for intracellular staining with FITC-conjugated RNF125 antibodies requires careful attention to several technical parameters:

• Fixation and Permeabilization:

  • For optimal results, use 4% paraformaldehyde for fixation (10-15 minutes at room temperature)

  • Test multiple permeabilization reagents (e.g., 0.1% Triton X-100, 0.1% saponin, or commercial permeabilization buffers) as RNF125 is predominantly intracellular

  • Gentle permeabilization is crucial to preserve epitope integrity while allowing antibody access

• Antibody Concentration and Incubation:

  • Titrate the FITC-conjugated RNF125 antibody to determine optimal concentration

  • Test extended incubation times (2-4 hours at room temperature or overnight at 4°C) to improve signal penetration

  • Include 1-2% BSA or serum in staining buffer to reduce non-specific binding

• Compensation Strategy:

  • FITC has potential spectral overlap with other fluorophores, particularly PE

  • Prepare single-stained controls for each fluorophore in your panel

  • If studying RNF125 in relation to PD-L1 or immune infiltrates, carefully design panels to avoid fluorophore combinations with significant overlap

• Signal Amplification Considerations:

  • For low-abundance RNF125 detection, consider a biotin-streptavidin system with FITC-streptavidin as a secondary step

  • Tyramide signal amplification can be employed for significantly enhanced sensitivity

• Experimental Validation:

  • Include RNF125 overexpression and knockdown controls to validate staining specificity

  • Use IFN-γ treated samples as positive controls, as this treatment affects RNF125-regulated pathways

This methodological approach is especially important when studying RNF125's relationship with immune checkpoint regulators or when attempting to correlate RNF125 expression with clinical outcomes in cancer research .

What troubleshooting strategies can address common issues with FITC photobleaching when imaging RNF125 in fixed specimens?

FITC photobleaching presents a significant challenge when imaging RNF125 in fixed specimens. Researchers can implement these evidence-based troubleshooting strategies:

• Anti-Fade Mounting Media Optimization:

  • Use specialized anti-fade mounting media containing anti-photobleaching agents

  • For long-term storage of specimens, mount with glycerol-based media containing n-propyl gallate (0.2-0.5%)

  • Commercial options with DABCO or PPD (p-phenylenediamine) provide superior protection for FITC fluorescence

• Imaging Parameter Adjustments:

  • Reduce excitation light intensity to 30-50% of maximum

  • Minimize exposure time while increasing camera gain or PMT sensitivity

  • Use narrowband FITC filters rather than broad spectrum filters to reduce unnecessary excitation

• Advanced Imaging Techniques:

  • Implement time-lapse protocols that capture images at intervals to reduce continuous exposure

  • For confocal microscopy, reduce laser power to <5% and increase pixel dwell time

  • Consider using resonant scanning confocal systems for faster acquisition with less photobleaching

• Chemical Photobleaching Reducers:

  • Add oxygen scavengers to mounting media (e.g., glucose oxidase/catalase system)

  • Include vitamin C (1-5 mM) in imaging buffer as a radical scavenger

  • For critical experiments, prepare specimens in an oxygen-depleted environment

• Signal Recovery Techniques:

  • For partially photobleached specimens, attempt signal recovery using low pH (5.0) PBS washes

  • Implement computational deconvolution to enhance signals from low-exposure images

These strategies are particularly important when attempting to visualize RNF125 co-localization with potential substrate proteins like PD-L1 or JAK1, where extended imaging sessions may be necessary to document protein-protein interactions .

How does RNF125's regulation of PD-L1 impact experimental design when studying cancer immunotherapy responses?

RNF125's newly discovered role as a regulator of PD-L1 significantly impacts experimental design in cancer immunotherapy research. Based on recent findings, researchers should consider these methodological implications:

• Expression Correlation Analysis:

  • When designing experiments to study immunotherapy resistance, researchers should systematically measure both RNF125 and PD-L1 levels, as they exhibit an inverse relationship

  • Flow cytometry protocols using FITC-conjugated RNF125 antibodies paired with differently labeled PD-L1 antibodies can quantify this relationship at the single-cell level

• Mechanistic Studies in Tumor Models:

  • Experimental designs should include both RNF125 knockout and overexpression models to fully characterize the impact on PD-L1 expression and tumor growth

  • The observation that RNF125 knockout MC-38 and H22 cells exhibited higher PD-L1 levels and faster tumor growth should inform control design in tumor challenge experiments

• Immune Infiltration Assessment:

  • Protocols should incorporate analysis of tumor-infiltrating lymphocytes alongside RNF125 quantification

  • The positive correlation between RNF125 expression and CD4+, CD8+ T cell and macrophage tumor infiltration suggests multiparameter flow cytometry or multiplexed immunofluorescence approaches are optimal

• Clinical Correlation Studies:

  • Patient stratification in clinical studies should consider RNF125 expression levels as a potential biomarker

  • The observation that RNF125's expression was downregulated in several human cancer tissues and negatively correlated with clinical stage supports this approach

• Therapeutic Response Prediction:

  • Experimental designs evaluating anti-PD-1/PD-L1 therapy should incorporate RNF125 expression analysis as a potential predictive biomarker

  • Studies should test whether restoring RNF125 expression can sensitize resistant tumors to checkpoint inhibition

This understanding is critical as it suggests that RNF125 expression levels could potentially predict responses to PD-1/PD-L1 blockade therapies, offering new avenues for stratifying patients and developing combination approaches .

What methodologies are appropriate for investigating RNF125's relationship with JAK1 signaling in melanoma resistance models?

Investigating RNF125's relationship with JAK1 signaling in melanoma resistance models requires specialized methodologies that can capture this complex regulatory interaction:

• Co-Immunoprecipitation and Ubiquitination Assays:

  • Implement Co-IP using FITC-conjugated RNF125 antibodies to visualize RNF125-JAK1 interactions directly

  • Design ubiquitination assays that specifically target K48-linked ubiquitination of JAK1 by RNF125

  • Include proteasome inhibitors like MG132 to stabilize interactions, as demonstrated in previous studies where endogenous RNF125-JAK1 interactions were detected only in the presence of MG132

• CRISPR/Cas9-Mediated Modulation:

  • Generate precise modifications in the RNF125 RING domain to disrupt E3 ligase activity without affecting protein-protein interactions

  • Create JAK1 mutants resistant to RNF125-mediated ubiquitination to establish causal relationships

  • Develop inducible RNF125 expression systems in BRAFi-resistant melanoma cells to study dynamic responses

• Receptor Tyrosine Kinase (RTK) Profiling:

  • Implement phospho-RTK arrays to comprehensively assess how RNF125 affects RTK expression through JAK1

  • Use the FITC-conjugated RNF125 antibody in conjunction with phospho-specific JAK1 antibodies to correlate RNF125 levels with JAK1 activation status

  • Monitor EGFR expression and activation as a specific downstream readout of the RNF125-JAK1 axis

• Combination Therapy Testing Platforms:

  • Develop high-throughput screening systems to test JAK1 and EGFR inhibitor combinations in melanoma models with varying RNF125 expression

  • Include patient-derived xenografts with known RNF125 status to validate findings from cell culture experiments

  • Implement in vivo imaging systems using fluorescently labeled antibodies to track therapy responses in real-time

• Transcriptional Regulation Analysis:

  • Investigate the Sox10/MITF regulatory axis in controlling RNF125 expression

  • Use ChIP-seq approaches to identify enhancer/promoter regions responsible for RNF125 downregulation in resistant melanomas

These methodologies provide a comprehensive framework for understanding how RNF125 downregulation contributes to BRAFi resistance through JAK1-mediated regulation of RTKs, potentially leading to new therapeutic strategies combining JAK1 and EGFR inhibition .

How can researchers effectively analyze correlations between RNF125 expression and immune cell infiltration in tumor microenvironments?

To effectively analyze correlations between RNF125 expression and immune cell infiltration in tumor microenvironments, researchers should implement these advanced methodological approaches:

• Multiplexed Immunofluorescence (mIF):

  • Design mIF panels incorporating FITC-conjugated RNF125 antibodies alongside markers for CD4+ T cells, CD8+ T cells, and macrophages

  • Implement cyclic immunofluorescence or spectral unmixing to overcome fluorophore limitations

  • Quantify spatial relationships between RNF125-expressing cells and immune infiltrates using computational image analysis

• Single-Cell RNA Sequencing (scRNA-seq) Integration:

  • Correlate RNF125 protein expression (measured by flow cytometry with FITC-conjugated antibodies) with scRNA-seq profiles

  • Develop computational pipelines to identify gene signatures associated with high vs. low RNF125 expression in both tumor and immune compartments

  • Analyze cell-cell communication networks between RNF125-expressing cells and infiltrating immune populations

• Spatial Transcriptomics:

  • Combine RNF125 immunodetection with spatial transcriptomics to correlate protein expression with gene expression signatures in situ

  • Map immune cell niches in relation to RNF125 expression gradients within the tumor microenvironment

  • This approach can validate and extend the positive correlation observed between RNF125 expression and immune cell infiltration

• Bioinformatic Analysis of Public Datasets:

  • Develop methodologies to mine TCGA data for correlations between RNF125 expression and immune infiltration signatures

  • Implement deconvolution algorithms to estimate immune cell abundances from bulk RNA-seq data

  • Create visualization tools to present multidimensional relationships between RNF125, PD-L1, and immune cell markers

• Experimental Manipulation and Longitudinal Imaging:

  • Design experimental systems with inducible RNF125 expression in tumor models

  • Implement intravital microscopy with fluorescently labeled immune cells to track infiltration dynamics in response to RNF125 modulation

  • Correlate temporal changes in RNF125 expression with immune recruitment patterns

These methodologies provide a comprehensive framework for analyzing the relationship between RNF125 expression and immune cell infiltration, building upon the finding that RNF125 expression positively correlates with CD4+, CD8+ T cell and macrophage tumor infiltration in both experimental models and public databases .

What experimental approaches are recommended for investigating RNF125's potential role in resistance to therapies beyond BRAF inhibitors?

Investigating RNF125's potential role in resistance to diverse cancer therapies requires sophisticated experimental approaches that build upon its established mechanisms:

• Therapy-Resistant Cell Line Panels:

  • Develop isogenic cell line panels with varied RNF125 expression levels and test resistance profiles against multiple targeted therapies (MEK inhibitors, immune checkpoint inhibitors, RTK inhibitors)

  • Use FITC-conjugated RNF125 antibodies for rapid screening of RNF125 expression in these models

  • Cross-reference resistance patterns with JAK1 and RTK activation status to identify common mechanisms

• CRISPR/Cas9 Resistance Screens:

  • Implement genome-wide CRISPR screens in the context of RNF125 overexpression or knockdown to identify synthetic lethal interactions

  • Focus on ubiquitination pathway components that might compensate for or synergize with RNF125

  • Use the identified genes to develop targeted combination strategies for resistant tumors

• Patient-Derived Models with Longitudinal Sampling:

  • Establish patient-derived organoids or xenografts from pre- and post-treatment samples

  • Track RNF125 expression changes during therapy using immunofluorescence with FITC-conjugated antibodies

  • Correlate expression patterns with treatment response and resistance development

• Signaling Network Analysis:

  • Apply phospho-proteomics to map signaling network alterations in RNF125-low versus RNF125-high settings

  • Focus on cross-talk between JAK/STAT, MAPK, and RTK pathways

  • Identify potential biomarkers of RNF125-mediated resistance that could be targeted in combination approaches

• Combined Immunotherapy Response Models:

  • Given RNF125's role in PD-L1 regulation, develop models testing sequential or concurrent administration of targeted therapies and immunotherapies

  • Monitor changes in the tumor immune microenvironment in relation to RNF125 expression

  • This approach builds on findings that RNF125 positively correlates with immune cell infiltration and may influence immunotherapy responses

These experimental approaches extend beyond the established role of RNF125 in BRAF inhibitor resistance to provide a comprehensive framework for understanding how this E3 ubiquitin ligase might influence responses to diverse cancer therapeutic strategies.

How can researchers most effectively use the FITC-conjugated RNF125 antibody to study dynamic changes in protein localization during cellular stress responses?

Studying dynamic changes in RNF125 localization during cellular stress requires sophisticated live-cell imaging approaches optimized for the FITC-conjugated antibody:

• Cell-Penetrating Antibody Modifications:

  • For live-cell applications, modify the FITC-conjugated RNF125 antibody with cell-penetrating peptides (CPPs) like TAT or Antennapedia

  • Optimize the CPP:antibody ratio to maximize cellular uptake while maintaining binding specificity

  • Validate that the modified antibody retains specificity for amino acids 143-231 of RNF125

• Stress Induction Protocols:

  • Design precise temporal stress induction systems (e.g., microfluidic devices for controlled application of stressors)

  • Implement hypoxia chambers with optical access for real-time imaging during oxygen deprivation

  • Develop protocols for UV irradiation or oxidative stress induction that are compatible with live-cell fluorescence imaging

• Advanced Imaging Modalities:

  • Utilize spinning disk confocal microscopy for high-speed acquisition with minimal phototoxicity

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to assess RNF125 mobility under different stress conditions

  • For super-resolution approaches, consider photo-switching strategies or expansion microscopy to visualize RNF125 association with subcellular structures

• Quantitative Image Analysis:

  • Develop computational pipelines for tracking RNF125 redistribution between cytoplasmic and nuclear compartments

  • Implement object-based colocalization analysis to quantify association with potential substrate proteins

  • Use machine learning approaches for pattern recognition in complex localization dynamics

• Multimodal Correlation:

  • Combine live imaging of FITC-RNF125 with sensors for ubiquitination activity

  • Correlate localization changes with functional readouts like JAK1 degradation or PD-L1 levels

  • This integrative approach provides mechanistic insights into how stress-induced relocalization affects RNF125's E3 ligase activity toward different substrates

These methodologies enable researchers to connect stress-induced changes in RNF125 localization with its functional roles in ubiquitination pathways, potentially revealing new regulatory mechanisms and therapeutic vulnerabilities.

Based on current research, what are the most promising future applications of RNF125 antibodies in translational research?

The most promising translational applications of RNF125 antibodies emerge from recent discoveries about its role in cancer and immune regulation:

• Precision Immunotherapy Biomarker Development:

  • RNF125 expression levels show significant potential as predictive biomarkers for immunotherapy response

  • FITC-conjugated antibodies enable multiparameter flow cytometry panels to simultaneously assess RNF125, PD-L1, and immune infiltration markers in patient samples

  • The negative correlation between RNF125 expression and clinical stage, coupled with better outcomes in patients with higher RNF125 expression, supports its development as a prognostic marker

• Companion Diagnostic Development:

  • For melanoma patients receiving BRAF inhibitors, RNF125 antibody-based diagnostics could identify patients likely to develop resistance

  • The relationship between RNF125 downregulation and JAK1/EGFR signaling suggests patients who might benefit from combination therapies targeting these pathways

  • Immunohistochemistry protocols using the antibody could be standardized for clinical laboratory implementation

• Therapeutic Target Validation:

  • As research progresses on restoring RNF125 expression or function, antibodies will be essential for validating target engagement

  • The antibody enables screening of small molecules that might stabilize RNF125 or enhance its E3 ligase activity toward specific substrates

  • Fluorescence-based high-content screening assays using FITC-conjugated antibodies can accelerate drug discovery efforts

• Monitoring Treatment Response:

  • Sequential liquid biopsies analyzed for circulating tumor cells with RNF125/PD-L1 quantification could provide real-time monitoring of treatment efficacy

  • Changes in RNF125 expression during treatment might serve as an early indicator of developing resistance

  • This application is supported by findings that RNF125 expression correlates with tumor stage and clinical outcomes

• Combination Therapy Rationale:

  • The mechanistic understanding of RNF125's role in regulating both PD-L1 and JAK1 provides strong rationale for combination approaches

  • The antibody enables preclinical validation of these combinations in patient-derived models

  • This is particularly relevant given evidence that JAK1 inhibition combined with EGFR inhibition overcame BRAFi resistance in melanoma with reduced RNF125 expression

These translational applications highlight the growing importance of RNF125 as both a biomarker and a key regulator at the intersection of targeted therapy resistance and immune checkpoint regulation.

What quality control measures should researchers implement when validating new lots of FITC-conjugated RNF125 antibodies?

Implementing rigorous quality control measures for FITC-conjugated RNF125 antibodies is essential for experimental reproducibility. Researchers should establish this comprehensive validation protocol:

• Spectroscopic Characterization:

  • Measure absorbance and emission spectra to confirm appropriate FITC conjugation

  • Calculate the fluorophore-to-protein ratio (typically 3-5 FITC molecules per antibody for optimal performance)

  • Monitor for any spectral shifts that might indicate suboptimal conjugation or degradation

• Binding Specificity Validation:

  • Perform side-by-side comparison with previous antibody lots using Western blotting and ELISA

  • Implement competitive binding assays with unconjugated antibody to confirm epitope specificity

  • Test against recombinant RNF125 protein fragments to verify recognition of the 143-231 amino acid region

• Cellular Validation Protocol:

  • Test in cell lines with confirmed RNF125 expression (positive control) and RNF125 knockout lines (negative control)

  • Compare staining patterns in immunofluorescence between lots

  • Verify expected subcellular localization patterns

  • Conduct flow cytometry on cells with manipulated RNF125 expression to confirm quantitative accuracy

• Functional Performance Metrics:

  Performance Parameter	Acceptance Criteria	Validation Method
  Signal-to-background ratio	≥5:1	Flow cytometry of RNF125-high vs. knockout cells
  Coefficient of variation	<15%	Repeated measurements of standard samples
  Lot-to-lot correlation	r² ≥0.9	Correlation analysis between current and reference lot
  Epitope recognition	>90% signal retention	Competitive binding with recombinant epitope
  FITC fluorescence stability	<10% signal loss over 4 hours	Continuous imaging under standard conditions

• Application-Specific Validation:

  • For researchers studying RNF125's role in PD-L1 regulation, verify the antibody can detect changes in RNF125 levels when PD-L1 expression is manipulated

  • For studies of JAK1 interaction, confirm the antibody doesn't interfere with the RNF125-JAK1 binding interface

  • When studying melanoma resistance mechanisms, validate performance in melanoma cell lines with varying BRAF inhibitor sensitivity