RNF34 Antibody

What is RNF34 and what cellular functions does it regulate?

RNF34 (Ring Finger Protein 34) is an E3 ubiquitin ligase that regulates several biological processes through ubiquitin-mediated proteasomal degradation of various target proteins. It plays critical roles in:

• Antiviral immunity: RNF34 binds to mitochondrial antiviral signaling protein (MAVS) following viral infection and negatively regulates RIG-I-like receptor (RLR)-mediated antiviral responses .

• Mitophagy: RNF34 is essential for clearance of damaged mitochondria after viral infection through catalyzing K27-/K29-linked ubiquitination of MAVS .

• Apoptosis regulation: RNF34 ubiquitinates caspases CASP8 and CASP10, promoting their proteasomal degradation and negatively regulating extrinsic apoptosis pathways .

• Brown fat metabolism: RNF34 mediates PPARGC1A proteasomal degradation, thereby regulating metabolism in brown fat cells .

• Cancer progression: RNF34 is upregulated in several cancer types including colorectal carcinomas and clear cell renal cell carcinoma (ccRCC) .

What applications are RNF34 antibodies most commonly used for?

RNF34 antibodies are primarily used in the following applications:

Many antibodies demonstrate reactivity with human samples, while some also cross-react with mouse, rat, and other species depending on the epitope targeted .

How should researchers validate RNF34 antibody specificity?

Rigorous validation of RNF34 antibodies should include:

• Knockdown verification: Compare antibody detection in control vs. RNF34 siRNA-treated cells. Research shows successful validation using shRNF34-1 and shRNF34-3 constructs that achieved >90% reduction in RNF34 expression .

• Peptide competition assay: Pre-incubation with the immunogenic peptide should abolish specific staining. This approach was used to validate rabbit anti-RNF34 antibodies in hippocampal neurons .

• Recombinant protein controls: Verify detection of purified His-RNF34 fusion proteins. Studies have used His-RNF34 (aa 195-381) fusion protein for this purpose .

• Overexpression verification: Compare immunofluorescence signals in transfected vs. non-transfected cells. Strong immunofluorescence signals were observed in HEK293 cells transfected with pCAGGS-RNF34 compared to neighboring non-transfected cells .

How can researchers study RNF34's role in antiviral immunity and MAVS ubiquitination?

To investigate RNF34's role in antiviral immunity, researchers should:

• Establish viral infection models: Studies have used vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV-GFP) to trigger RIG-I pathways and RNF34 activity .

• Analyze ubiquitination patterns: Specifically examine K27-/K29-linked ubiquitination of MAVS at lysine residues 297, 311, 348, and 362, which serve as recognition signals for NDP52-dependent autophagic degradation .

• Track ubiquitination transitions: Investigate how RNF34 mediates the K63- to K27-linked ubiquitination transition on MAVS (primarily at Lys 311), facilitating autophagic degradation upon RIG-I stimulation .

• Perform co-immunoprecipitation assays: Use RNF34 antibodies to capture protein complexes including MAVS and RIG-I. This approach revealed that RNF34 interacts with both proteins but primarily reduces MAVS levels .

• Assess downstream signaling effects: Measure TBK1 and IRF3 phosphorylation, IFN-β production, and expression of ISGs (ISG54, ISG56) in control versus RNF34-knockdown cells following viral infection .

What methodological approaches can be used to investigate RNF34 in mitophagy?

To study RNF34's role in mitophagy, researchers can employ these techniques:

• Track mitochondrial protein degradation: Monitor levels of constitutive mitochondria-localized proteins (TOM20, HSP60) via immunoblotting to assess mitophagy in control versus RNF34-deficient cells .

• Employ fluorescent mitophagy reporters: Use pH-sensitive fluorescent proteins like Keima targeted to mitochondria. RNF34 overexpression stimulates a shift in Keima fluorescence from 458 to 543 nm, indicating increased lysosomal translocation of mitochondria during viral infection .

• Utilize transmission electron microscopy: Directly observe mitochondrial clearance defects in RNF34-knockdown cells in response to viral infection .

• Perform co-localization studies: Analyze co-localization between TOM20 and LAMP1 or between NDP52 and MAVS in the presence or absence of RNF34 to assess mitophagy progression .

• Control for apoptosis effects: Use Z-VAD-FMK (pan-caspase inhibitor) to block virus-induced cell death and isolate mitophagy-specific effects when studying mitochondrial clearance .

How can RNF34 antibodies be employed in cancer research and prognostic studies?

RNF34 antibodies have significant applications in cancer research:

For nuclear expression specifically:

For progression-free survival (membranous expression):

These findings suggest RNF34 is an independent prognostic biomarker for ccRCC .

What are the key considerations for optimizing western blot protocols with RNF34 antibodies?

For optimal western blot results with RNF34 antibodies:

• Sample preparation: Use lysis buffers containing protease inhibitors. Some studies used buffers containing 10 μM MG132 to prevent proteasomal degradation during sample preparation .

• Expected molecular weight: Detect RNF34 at approximately 42 kDa, its calculated molecular weight . Some antibodies may detect RNF34 at apparent molecular weights up to 68 kDa depending on post-translational modifications .

• Recommended dilutions: Most commercial RNF34 antibodies work optimally at dilutions between 1:500-1:5000 for western blotting .

• Blocking conditions: Standard blocking with 5% non-fat milk in TBST is typically sufficient, though some protocols may benefit from specialized blocking reagents.

• Controls: Include positive control lysates from cell lines known to express RNF34 (U-251MG, LO2, HeLa, BT-474) .

How does RNF34 expression change during development and in response to stimuli?

RNF34 expression is dynamically regulated:

• Developmental regulation: Rabbit anti-RNF34 antibodies have been used to study RNF34 expression during brain development, demonstrating temporal and spatial expression patterns .

• Cold-induced regulation: RNF34 is a cold-regulated E3 ubiquitin ligase in brown adipose tissue. Mice placed at 4°C for 5 hours showed altered RNF34 expression in brown fat compared to those at room temperature .

• β-adrenergic stimulation: Treatment with CL-316243 (β3-adrenergic receptor agonist, 0.5 mg/kg body weight, daily for 6 days) affects RNF34 expression in brown and white adipose tissue .

• Viral infection: RNF34 is upregulated and translocates to the mitochondrial outer membrane (where MAVS resides) in response to viral infection .

What methodological approaches can be used to study RNF34's E3 ligase activity?

To investigate RNF34's E3 ligase function:

• Ubiquitination assays: Compare wild-type RNF34 with RNF34 H342A E3 ligase-dead mutant in ubiquitination experiments. The H342A mutation compromises inhibition of VSV-mediated IFN-β and NF-κB activation .

• Linkage-specific ubiquitin analysis: Use antibodies specific for different ubiquitin linkages (K27-, K29-, K48-, K63-linked) to determine the precise ubiquitin chains catalyzed by RNF34 on target proteins .

• Ubiquitination site mapping: Analyze lysine mutants of target proteins (e.g., MAVS K311R) to identify specific ubiquitination sites. Research showed RNF34 primarily targets MAVS at lysine residues 297, 311, 348, and 362 .

• Co-immunoprecipitation: Use RNF34 antibodies to pull down protein complexes and identify potential substrates or cofactors in the ubiquitination process .

• In vitro reconstitution: Reconstitute the ubiquitination reaction with purified components including E1, E2, RNF34, ubiquitin, and substrate proteins to directly assess E3 ligase activity.

How might RNF34 serve as a therapeutic target in cancer and viral diseases?

Given RNF34's roles in cancer progression and antiviral immunity, several therapeutic approaches warrant investigation:

What emerging techniques could enhance RNF34 research?

Future RNF34 research could benefit from these advanced methodologies:

• CRISPR/Cas9 genome editing: Generate precise RNF34 knockout or knock-in models to study function in various tissues and disease models.

• Proximity labeling: Use BioID or APEX2 fused to RNF34 to identify proximal proteins and potential novel substrates in living cells.

• Single-cell analysis: Examine RNF34 expression and function at single-cell resolution to understand heterogeneity in cancer and immune responses.

• Structural studies: Determine the three-dimensional structure of RNF34 alone and in complex with substrates to guide rational drug design.

• Super-resolution microscopy: Track RNF34 localization during cellular processes with nanometer precision, particularly during viral infection and mitophagy.

• Proteomics approaches: Use quantitative proteomics to identify the complete set of proteins regulated by RNF34-mediated ubiquitination under different conditions.

• Animal models: Develop tissue-specific RNF34 knockout mice to better understand its physiological roles in development and disease.