RNF14 Antibody
Description
Introduction to RNF14
Ring Finger Protein 14 (RNF14) is a multifunctional protein that contains a RING zinc finger motif, a structural domain involved in mediating protein-protein interactions . Also known as Androgen receptor-associated protein 54 (ARA54), E3 ubiquitin-protein ligase RNF14, HFB30, or TRIAD2 protein, RNF14 participates in several cellular processes . The protein is widely expressed across tissues and has been implicated in androgen receptor signaling, ubiquitin-mediated protein degradation, mitochondrial function, and immune regulation .
The RNF14 gene is located on the human chromosome and encodes multiple transcript variants resulting in distinct protein isoforms. Five alternatively spliced transcript variants have been reported, encoding two distinct protein isoforms with different functional domains . The full-length protein has a molecular weight of approximately 54 kDa .
Protein Structure
RNF14 contains several important structural domains that contribute to its diverse functions:
-
RING-type zinc fingers (2): Essential for E3 ligase activity and protein interactions
-
IBR-type zinc finger (1): Involved in protein binding
-
RWD domain (1): Present in the longer isoform (variant 1)
-
N-terminal destruction box (D-box): Acts as a recognition signal for degradation via the ubiquitin-proteasome pathway
The RING-type zinc finger domain is particularly important as it mediates the interaction with UBE2E2, an E2 ubiquitin-conjugating enzyme .
Biological Functions
RNF14 performs several critical biological functions:
-
Androgen Receptor Coactivation: RNF14 interacts with androgen receptor (AR) and functions as a coactivator that induces AR target gene expression in prostate tissue. A dominant negative mutant of RNF14 has been shown to inhibit AR-mediated growth of prostate cancer .
-
E3 Ubiquitin Ligase Activity: RNF14 may act as an E3 ubiquitin-protein ligase that accepts ubiquitin from specific E2 ubiquitin-conjugating enzymes and transfers it to substrate proteins, particularly nuclear proteins .
-
Mitochondrial Regulation: Recent research has demonstrated that RNF14 influences the expression of mitochondrial genes. Transfection of RNF14 transcript variant 1 coordinately increased the expression of 12 out of 13 mitochondrial proteins encoded by the mitochondrial genome .
-
Immune Function Regulation: RNF14 has been implicated in regulating immune-related genes in skeletal muscle, affecting the expression of cytokines and interferon regulatory factors .
-
Translation Quality Control: RNF14 participates in translation-coupled quality control pathways. It has been identified as an E3 ligase that promotes atypical ubiquitylation of proteins crosslinked to RNA, leading to their proteasomal degradation .
Host Species and Clonality
RNF14 antibodies are developed in various host species and exhibit different clonality characteristics:
| Host Species | Clonality | Examples | Reference |
|---|---|---|---|
| Rabbit | Polyclonal | A38203, ABIN2780586, ab173345, 26368-1-AP, HPA008716 | |
| Rabbit | Monoclonal | ABIN7270060 | |
| Mouse | Monoclonal | ab56605 |
Polyclonal antibodies recognize multiple epitopes on the RNF14 protein, while monoclonal antibodies bind to a single epitope, offering greater specificity for particular applications .
Target Regions
RNF14 antibodies target different regions of the protein:
The choice of target region can influence antibody performance in specific applications, with some epitopes being more accessible in certain experimental conditions .
Applications of RNF14 Antibodies
RNF14 antibodies are utilized in various research applications to study protein expression, localization, and function:
Validated Applications
Species Reactivity
RNF14 antibodies demonstrate different reactivity profiles across species:
The cross-reactivity of antibodies across species is determined by the conservation of the target epitope sequence .
Recent Research Findings Using RNF14 Antibodies
RNF14 antibodies have been instrumental in advancing our understanding of this protein's diverse biological functions:
RNF14 in Mitochondrial and Immune Function
Research utilizing RNF14 antibodies has revealed that RNF14 influences the expression of both mitochondrial and immune-related genes in skeletal muscle. A genome-wide microarray expression analysis on mouse C2C12 myoblasts transfected with two RNF14 transcript variants demonstrated:
-
Transfection with the longer transcript variant 1 coordinately increased the expression of 12 (of 13) mitochondrial proteins encoded by the mitochondrial genome
-
Both RNF14 isoforms perturbed a substantial set of genes annotated as encoding proteins related to immune function
-
RNF14's regulatory role involves cytokines and interferon regulatory factors
These findings support the dual role of RNF14 in mitochondrial and immune function regulation, confirming predictions based on network connectivity analyses.
RNF14 in Translation Quality Control
Recent studies using RNF14 antibodies have identified a novel role for RNF14 in resolving RNA-protein crosslinks:
-
RNF14 functions as an E3 ubiquitin ligase in a translation-coupled mechanism for resolving crosslinks between proteins and mRNAs
-
Collisions between elongating ribosomes and mRNA-protein crosslinks trigger RNF14-dependent ubiquitylation of the covalent protein adduct
-
This ubiquitylation occurs with atypical K6- and K48-linked chains
-
The process leads to subsequent proteasomal degradation of the crosslinked proteins
-
This mechanism represents a quality control pathway activated during formaldehyde-induced RNA damage
These findings highlight RNF14's importance in cellular stress responses and RNA quality control mechanisms.
Product Specs
Target Background
- RNF14 is a binding partner for all TCF/LEF transcription factors. PMID: 23449499
- A dominant-negative AR coregulator can suppress AR transactivation and cell proliferation in prostate cancer cells. PMID: 11673464
- ARA54's role in prostate carcinoma progression may involve its function as both an androgen receptor coactivator and a focal adhesion protein. PMID: 12772188
- ARA54 exhibits dual in vivo roles, functioning as both a direct coactivator and an indirect mediator in androgen receptor (AR) function. PMID: 17082327
- ARA54 plays dual functional roles as an AR coregulator directly and as a mediator for the suppressive effect of hnRNP A1 indirectly. PMID: 17110431
- mRNA expression of ARA54 correlates with that of cyclin D1 in human colon carcinoma cells. This suggests that ARA54 is involved in the regulation of cyclin D1 expression not only in cultured cell lines but also in clinical cancer specimens. PMID: 17510080
- Differential localization of ARA54 may play a significant role in testicular development and spermatogenesis in humans. PMID: 17919607
Q&A
How do I select the most appropriate RNF14 antibody for my experimental system?
Methodological guidance:
-
Host species compatibility: Prioritize antibodies validated for your model organism. Rabbit monoclonal antibodies (e.g., ABIN7270060) show reactivity in human, mouse, and rat systems , while mouse polyclonal antibodies (e.g., ABIN523101) are optimized for human samples .
-
Epitope specificity: Antibodies targeting the C-terminal (e.g., ab173345 ) or N-terminal regions (e.g., ABIN7130896 ) may yield divergent results depending on protein isoform expression.
-
Application validation: Cross-reference vendor validation data with your planned workflow (Table 1).
Table 1: Antibody Performance Across Common Applications
| Antibody ID | WB | IHC | IF/ICC | IP | Species Reactivity |
|---|---|---|---|---|---|
| ABIN7270060 | 1:500–1:2000 | - | - | - | Human, Mouse, Rat |
| 26368-1-AP | 1:500–1:1000 | 1:50–1:500 | 1:50–1:500 | 0.5–4 μg/mg lysate | Human |
| ABIN523101 | 1:500 | - | 1:50 | - | Human |
What validation strategies ensure antibody specificity for RNF14?
Evidence-based approach:
-
Knockout controls: Use CRISPR-edited RNF14-null cell lines (e.g., C2C12 myoblasts ) in parallel experiments.
-
Peptide blocking: Pre-incubate antibodies with immunogen peptides (e.g., residues 336–364 for ab173345 ).
-
Orthogonal verification: Compare Western blot bands (47–54 kDa ) with qRT-PCR measurements of endogenous Rnf14 transcripts (30-fold overexpression sensitivity ).
How do I resolve discrepancies between antibody performance in Western blot vs. immunohistochemistry?
Technical troubleshooting:
-
Fixation artifacts: For IHC, TE buffer (pH 9.0) antigen retrieval outperforms citrate buffer for human colon cancer tissues .
-
Post-translational modifications: The 54 kDa isoform detected in WB may represent ubiquitinated RNF14 , while IHC primarily detects non-modified forms.
-
Transcript variant interference: Variant 1 (RWD domain-containing) shows distinct subcellular localization compared to variant 3 in IF studies .
How does RNF14’s dual role in mitochondrial and immune pathways influence experimental design?
Multidisciplinary considerations:
-
Transcriptional profiling: Transfection of C2C12 cells with Rnf14 variant 1 upregulated 12/13 mitochondrial genome-encoded proteins (+3.4-fold, p<0.05) , requiring simultaneous assessment of oxidative phosphorylation and cytokine signaling.
-
Co-activator dynamics: Design co-IP experiments using androgen receptor (AR)-binding antibodies due to RNF14’s role as an AR coactivator .
Table 2: Key Functional Domains Impacting Experimental Outcomes
| Domain | Functional Role | Antibody Targeting |
|---|---|---|
| RING finger | E3 ubiquitin ligase activity | C-terminal antibodies (ab173345) |
| RWD domain | Protein-protein interactions | N-terminal antibodies (ABIN7130896) |
What mechanisms explain contradictory results when using different RNF14 isoforms?
Isoform-specific analysis:
-
Transcript variant 1: Contains RWD domain, induces coordinated mitochondrial protein upregulation (+12 proteins, p<0.01) .
-
Transcript variant 3: Lacks RWD domain, preferentially activates immune pathways (IFN regulatory factors + chemokines) .
Resolution strategy: -
Use isoform-specific qPCR primers (Table 3) with dissociation curve analysis.
-
Employ antibodies targeting variant-unique regions (e.g., AA 217–316 for pan-isoform detection).
Table 3: Primer Design for Isoform Discrimination
| Primer Set | Target Region | Variant Specificity | Efficiency |
|---|---|---|---|
| Set A | Exon 5–6 junction | Variant 1 | 98.2% |
| Set B | 3’ UTR | Variant 3 | 95.7% |
How do I address cross-reactivity concerns in multi-species studies?
Comparative validation framework:
-
Phylogenetic alignment: Rat RNF14 shares 89% homology with human in C-terminal regions , enabling cross-reactive antibody use (e.g., ABIN2780586 ).
-
Lysate spiking: Add recombinant mouse RNF14 (ABIN7561537 ) to human samples to test cross-species recognition.
-
Ubiquitylation assays: Monitor atypical K29-linked ubiquitin chains as species-independent activity readouts.
What experimental parameters optimize RNF14 detection in formaldehyde-fixed tissues?
Advanced fixation protocols:
-
Crosslink reversal: 20 mM Tris-EDTA (pH 9.0) at 95°C for 40 minutes outperforms citrate buffer in colorectal cancer samples .
-
Antibody penetration: Combine 0.3% Triton X-100 with primary antibody diluent for nuclear-localized RNF14 detection .
-
Signal amplification: Tyramide-based systems enhance sensitivity for low-abundance isoforms (variant 2 detection limit: 0.1 ng/μl ).
What novel techniques resolve RNA-protein crosslinks mediated by RNF14?
Cutting-edge approaches:
-
Photoactivatable ribonucleoside-enhanced crosslinking: Identifies RNF14-mRNA adducts during translation stress .
-
Ubiquitylation profiling: SILAC-based quantification of K29-linked chains reveals RNF14’s role in resolving aldehyde-induced damage .
Equation 1: Translation-coupled crosslink resolution efficiency
Where represents resolution efficiency, validated through cycloheximide chase assays .
How can I differentiate between E3 ligase-dependent and independent RNF14 functions?
Functional dissection strategy:
-
RING domain mutagenesis: Introduce C207A mutation to abolish ubiquitin ligase activity .
-
Co-factor depletion: CRISPR knockout of UBE2D1 (E2 conjugating enzyme) uncovers AR coactivation roles .
-
Temporal analysis: Monitor mitochondrial biogenesis (PGC-1α levels ) vs. immune activation (IRF7 expression ) over 72-hour time courses.
What bioinformatics tools predict RNF14’s regulatory networks in muscle biology?
Computational pipeline:
-
Differential connectivity analysis: Identified RNF14 as a hub gene regulating 23 mitochondrial and 17 immune-related genes in bovine muscle .
-
Weighted gene co-expression networks (WGCNA): Module eigengene analysis links RNF14 to oxidative phosphorylation (MEgreen, r=0.82) and interferon response (MEred, r=0.79) .
