RNF125 Antibody, HRP conjugated

What is RNF125 and why is it important to study?

RNF125 functions as an E3 ubiquitin-protein ligase that mediates ubiquitination and subsequent proteasomal degradation of target proteins including RIG-I, MAVS/IPS1, IFIH1/MDA5, JAK1, p53/TP53, and PD-L1 . It plays critical roles in regulating T-cell activation, cancer immunotherapy responses, and innate immune function. Recent studies have identified RNF125 as a negative regulator of PD-L1, highlighting its potential significance in cancer immunotherapy approaches . Additionally, downregulation of RNF125 has been associated with resistance to BRAF inhibitors in melanoma, making it relevant to cancer therapy resistance mechanisms .

What detection methods can be used with HRP-conjugated RNF125 antibodies?

HRP-conjugated antibodies enable direct enzyme-linked detection without requiring secondary antibodies. For RNF125 detection, Western blotting is the most validated application based on available antibodies . When using HRP-conjugated primary antibodies, researchers should employ chemiluminescent, colorimetric, or fluorescent substrates compatible with horseradish peroxidase. Importantly, Western blot analysis of RNF125 typically reveals bands at both the predicted size (26 kDa) and occasionally at 100 kDa, which may represent post-translationally modified forms or protein complexes . Enhanced chemiluminescence (ECL) substrates are particularly effective for detecting low-abundance proteins like RNF125.

How should samples be prepared for optimal RNF125 detection?

When preparing samples for RNF125 detection, optimal lysis buffer selection is crucial. RIPA buffer supplemented with protease inhibitors effectively extracts both membrane-associated and cytosolic RNF125. Sample preparation should include reducing conditions as demonstrated in validated protocols . Since RNF125 regulates protein degradation pathways, treating cells with proteasome inhibitors (e.g., MG132) before lysis can enhance detection of ubiquitinated substrates and potentially RNF125 itself. This approach was demonstrated in studies examining RNF125's interaction with its substrates, where MG132 treatment enabled detection of endogenous RNF125-JAK1 interactions .

How can I verify RNF125 antibody specificity in ubiquitination assays?

Verifying RNF125 antibody specificity in ubiquitination assays requires multiple complementary approaches. The gold standard approach combines genetic knockout/knockdown controls with overexpression systems. Researchers should generate RNF125 knockdown or knockout cell lines using shRNA or CRISPR-Cas9, respectively, which should show diminished antibody signal . Additionally, overexpression of both wild-type RNF125 and catalytically inactive RING mutant (as used in published studies) can distinguish between specific and non-specific signals .

For ubiquitination assays specifically, immunoprecipitation followed by immunoblotting with anti-ubiquitin antibodies can confirm RNF125's E3 ligase activity. Studies have successfully employed this approach to demonstrate RNF125-mediated ubiquitination of substrates like PD-L1 and JAK1 . Including appropriate controls such as the RING domain mutant, which lacks E3 ligase activity, strengthens the specificity assessment of these experiments.

What are the optimal conditions for studying RNF125-mediated ubiquitination of PD-L1?

Studying RNF125-mediated ubiquitination of PD-L1 requires careful experimental design. Co-transfection experiments in HEK293T cells have successfully demonstrated this interaction using tagged constructs (HA-RNF125 and Flag-PD-L1) . For detecting endogenous interactions, co-immunoprecipitation experiments in cell lines with detectable levels of both proteins (such as HepG2) have been effective .

The ubiquitination assay should include MG132 treatment (5-10 μM for 4-6 hours) to prevent proteasomal degradation of ubiquitinated proteins. Western blot analysis should specifically examine K48-linked polyubiquitination, as this linkage type mediates PD-L1 degradation by RNF125 . For functional validation, comparing PD-L1 protein half-life in the presence and absence of RNF125 using cycloheximide chase experiments provides crucial evidence of RNF125's effect on PD-L1 stability. Researchers should also consider IFN-γ treatment conditions, as PD-L1 levels are responsive to this cytokine, and RNF125 has been shown to regulate PD-L1 even under IFN-γ stimulation .

How can I investigate the role of RNF125 in BRAF inhibitor resistance using antibody-based approaches?

Investigating RNF125's role in BRAF inhibitor resistance requires comparative analysis between sensitive and resistant cell populations. Researchers should establish BRAF inhibitor-resistant cell lines (e.g., through chronic exposure to increasing concentrations of BRAF inhibitors) and parental sensitive lines . Western blot analysis using RNF125 antibodies can then quantitatively assess expression differences between these populations.

For mechanistic studies, examining JAK1 levels is crucial as JAK1 is a key substrate of RNF125 and is associated with resistance mechanisms . Complementary gain-of-function and loss-of-function experiments should be performed, with re-expression of wild-type RNF125 (but not the RING mutant) in resistant cells to determine if restoring RNF125 function resensitizes cells to BRAF inhibitors . Monitoring of receptor tyrosine kinase (RTK) expression patterns, which are influenced by RNF125 via JAK1 regulation, provides additional mechanistic insights. Growth assays in both 2D and 3D culture systems have demonstrated measurable phenotypic differences that correlate with RNF125 expression status .

Why might I observe multiple bands when detecting RNF125 by Western blot?

The detection of multiple bands when probing for RNF125 by Western blot is a common technical challenge. Published data shows bands at both 26 kDa (predicted molecular weight) and 100 kDa . Multiple factors could explain this observation:

• Post-translational modifications: As an E3 ubiquitin ligase, RNF125 may undergo auto-ubiquitination or other modifications

• Protein complexes: Incomplete sample denaturation may preserve RNF125-containing complexes

• Alternative splice variants: Different isoforms may exist in certain cell types

• Cross-reactivity: Antibodies may detect related RING finger proteins

To determine the specific band representing RNF125, researchers should implement controls including RNF125 knockdown/knockout samples and overexpression of tagged RNF125 constructs. Antibody validation using multiple RNF125 antibodies targeting different epitopes can help identify the authentic signal. The observation of a 100 kDa band in published Western blots suggests this may represent a biologically relevant form of RNF125 rather than non-specific binding .

What factors might affect RNF125 antibody sensitivity in different experimental contexts?

Several factors can influence RNF125 antibody sensitivity across experimental systems. First, endogenous RNF125 expression levels vary significantly between cell types and can be particularly low in some contexts, requiring signal amplification strategies. RNF125 expression has been shown to be downregulated in several human cancer tissues compared to normal tissues .

Second, RNF125 protein stability is regulated by its own ubiquitin ligase activity (auto-ubiquitination), so proteasome inhibitors like MG132 can enhance detection by preventing degradation . Third, the epitope accessibility may be affected by protein-protein interactions or post-translational modifications, particularly in the context of different cellular signaling states.

For optimal sensitivity, researchers should consider these specific optimization strategies:

• Enhanced chemiluminescent substrates with longer exposure times

• Sample enrichment through immunoprecipitation before Western blotting

• Signal amplification using biotin-streptavidin systems for immunohistochemistry applications

• Careful selection of lysis buffers that efficiently extract RNF125 while preserving epitope integrity

How can I resolve discrepancies between RNF125 mRNA and protein expression data?

Discrepancies between RNF125 mRNA and protein levels are commonly observed due to post-transcriptional regulation mechanisms. Studies have reported instances where JAK1 protein levels increased in BRAF inhibitor-resistant cells without corresponding changes in mRNA levels, suggesting post-transcriptional regulation via RNF125-mediated protein degradation .

To investigate such discrepancies:

• Perform parallel qRT-PCR and Western blot analyses on the same samples to directly compare transcript and protein levels

• Assess protein stability using cycloheximide chase experiments, which have revealed extended JAK1 half-life in cells with reduced RNF125 expression

• Employ proteasome inhibitors (MG132) to determine if protein levels equalize when degradation is blocked

• Consider translational regulation by analyzing polysome-associated RNF125 mRNA

When publishing these findings, clearly present both mRNA and protein data with appropriate statistical analyses to highlight the discrepancies and their potential biological significance. This approach has successfully elucidated the post-transcriptional regulatory mechanisms controlling RNF125 substrates in previous studies .

How does RNF125 expression correlate with clinical outcomes in cancer patients?

RNF125 expression has significant correlations with clinical outcomes in multiple cancer types. Analysis of The Cancer Genome Atlas (TCGA) database has revealed that RNF125 expression is significantly downregulated in several human cancer tissues compared to normal tissues . Additionally, RNF125 expression levels negatively correlate with clinical stage in multiple cancer types, with lower expression associated with more advanced disease .

Survival analysis indicates that patients with higher RNF125 expression generally have better clinical outcomes . This favorable prognostic value may be linked to RNF125's role in regulating immune checkpoints, particularly PD-L1. Mechanistically, higher RNF125 expression leads to increased degradation of PD-L1, potentially enhancing anti-tumor immune responses .

Furthermore, RNF125 expression positively correlates with CD4+ T cell, CD8+ T cell, and macrophage tumor infiltration, suggesting its involvement in modulating the tumor immune microenvironment . These findings collectively indicate that RNF125 expression analysis may have value as a prognostic biomarker and could potentially predict responsiveness to immunotherapy approaches.

How can I integrate RNF125 data with other ubiquitin ligase expression patterns?

Integrating RNF125 data with other ubiquitin ligases requires systematic bioinformatic approaches. First, researchers should curate a comprehensive list of E3 ubiquitin ligases that target overlapping substrates or function in related pathways. For RNF125, this would include other ubiquitin ligases known to regulate PD-L1 (such as STUB1/CHIP), JAK1, or p53.

Network analysis approaches can then identify functional relationships between these ubiquitin ligases. Protein-protein interaction databases and pathway enrichment analyses help establish the biological context of these relationships. Gene expression correlation analysis across tissue types or disease states can reveal coordinated or compensatory regulation patterns among E3 ligases.

For experimental validation, researchers can perform combinatorial knockdown/knockout experiments to identify functional redundancy or synergy. When analyzing patient samples, multiplexed immunohistochemistry or proteomics approaches allow simultaneous detection of multiple ubiquitin ligases. These integrated analyses provide deeper insights into the ubiquitin-proteasome network's role in disease processes and potential therapeutic opportunities.

What is the significance of RNF125-mediated regulation of immune checkpoint molecules?

RNF125-mediated regulation of immune checkpoint molecules, particularly PD-L1, has profound implications for cancer immunotherapy. As demonstrated in recent studies, RNF125 interacts directly with PD-L1 and negatively regulates its expression through K48-linked polyubiquitination, which targets PD-L1 for proteasomal degradation . This regulatory mechanism represents a novel post-translational control point for this critical immune checkpoint molecule.

The biological significance of this regulation is evident from tumor growth studies. RNF125 knockout in tumor cell lines (MC-38 and H22) resulted in higher PD-L1 levels and accelerated tumor growth in mouse models, while RNF125 overexpression had the opposite effect, reducing PD-L1 levels and slowing tumor growth . Importantly, immunohistochemical analysis revealed that tumors with RNF125 overexpression exhibited significantly increased infiltration of CD4+ T cells, CD8+ T cells, and macrophages, indicating enhanced anti-tumor immune responses .

These findings suggest that therapeutic approaches that upregulate or stabilize RNF125 could potentially enhance the efficacy of existing PD-1/PD-L1 blockade immunotherapies. Conversely, understanding RNF125 expression patterns in patient tumors could help identify individuals more likely to respond to checkpoint inhibitor therapies. This regulatory axis represents an important consideration for the development of next-generation cancer immunotherapy strategies.

How might RNF125 antibodies be utilized in developing combination therapy approaches?

RNF125 antibodies can serve as crucial tools for developing and optimizing combination therapy approaches. Given that RNF125 downregulation contributes to BRAF inhibitor resistance in melanoma through JAK1 stabilization and subsequent receptor tyrosine kinase (RTK) upregulation, monitoring RNF125 and JAK1 levels using antibody-based assays can guide rational drug combinations .

Preclinical studies have already demonstrated that combined inhibition of JAK1 and EGFR can overcome BRAF inhibitor resistance in melanomas with reduced RNF125 expression . RNF125 antibodies enable patient stratification through immunohistochemical analysis of tumor samples, identifying patients with low RNF125 expression who might benefit from specific combination approaches.

For immunotherapy applications, RNF125's regulation of PD-L1 suggests potential synergies between agents that modulate RNF125 expression/activity and checkpoint inhibitors . Utilizing RNF125 antibodies to monitor treatment-induced changes in RNF125 levels could provide pharmacodynamic biomarkers for such combination strategies. Furthermore, high-throughput screening approaches employing RNF125 antibodies could identify novel compounds that increase RNF125 expression or enhance its E3 ligase activity toward specific substrates like PD-L1 or JAK1.

What novel approaches could enhance the sensitivity of RNF125 detection in clinical samples?

Enhancing RNF125 detection sensitivity in clinical samples requires innovative technical approaches. Proximity ligation assays (PLA) offer superior sensitivity for detecting protein-protein interactions, such as RNF125 with its substrates. This approach could visualize RNF125-PD-L1 or RNF125-JAK1 complexes in situ within tissue sections, providing spatial context that is impossible with traditional biochemical methods.

Digital pathology platforms combined with machine learning algorithms can improve quantification of RNF125 immunohistochemistry, particularly in heterogeneous tissue samples. These approaches can better distinguish subtle differences in expression patterns that may have clinical significance.

Mass spectrometry-based approaches, particularly selected reaction monitoring (SRM) or parallel reaction monitoring (PRM), offer peptide-level quantification of RNF125 and its post-translational modifications. This approach circumvents antibody limitations and can distinguish between RNF125 isoforms or modified forms.

Single-cell technologies could reveal RNF125 expression heterogeneity within tumors and correlate this with other markers of immune activity or drug resistance. Antibody-based enrichment prior to single-cell analysis can enhance detection of low-abundance proteins like RNF125. These advanced approaches will likely provide more comprehensive insights into RNF125 biology in clinical contexts.

How can RNF125 substrates be systematically identified and validated?

Systematic identification and validation of RNF125 substrates requires integrated proteomic and genetic approaches. Initial substrate screening can employ mass spectrometry-based proteomics comparing ubiquitinated proteins in control versus RNF125-depleted or overexpressing cells. Previous research successfully used LC-MS/MS analysis with the RING mutant form of RNF125 to identify JAK1 as a substrate .

For candidate validation, researchers should demonstrate:

• Physical interaction between RNF125 and the candidate substrate through co-immunoprecipitation assays, as demonstrated for PD-L1 and JAK1

• RNF125-dependent ubiquitination using in vitro and cellular ubiquitination assays

• Regulation of substrate stability through cycloheximide chase experiments comparing wild-type to RNF125-deficient conditions

• Rescue experiments showing that catalytically inactive RNF125 (RING mutant) fails to regulate the substrate