rnf44 Antibody

What is RNF44 and what experimental approaches should be used to study its biological functions?

RNF44 (RING finger protein 44) is an E3 ubiquitin ligase that plays important roles in substrate recognition and protein degradation through the ubiquitin-proteasome system. The protein contains characteristic RING finger domains that are essential for its E3 ligase activity .

Recommended methodological approaches:

• Bioinformatic analysis of RNF44 expression across normal and disease tissues using GTEx and TCGA databases

• RNF44 knockdown using shRNA plasmids to evaluate function in cellular models

• Co-immunoprecipitation to identify RNF44 binding partners and substrates

• Ubiquitination assays to validate E3 ligase activity on specific targets

• Correlation analysis between RNF44 expression and clinical parameters

Based on existing research, RNF44 has been implicated in hepatocellular carcinoma (HCC) progression, with overexpression correlating with poor prognosis and immune cell infiltration patterns .

What validation methods should be employed when using RNF44 antibodies in experimental settings?

Recommended validation protocol:

• Western blot analysis using recombinant RNF44 control fragment (such as aa 1-88) alongside endogenous samples

• Peptide competition assay: Pre-incubate antibody with 100x molar excess of the control peptide for 30 minutes at room temperature before application in IHC/ICC or WB

• siRNA/shRNA knockdown controls to confirm specificity of antibody signal

• Cross-validation using antibodies targeting different epitopes of RNF44

• Include negative control tissues known to have low RNF44 expression based on RNA-seq data

Important considerations: The Human Protein Atlas has validated some commercially available RNF44 antibodies in multiple tissues, providing a benchmark for expected staining patterns .

What technical considerations are crucial for optimizing RNF44 immunostaining protocols?

When performing IHC or ICC experiments with anti-RNF44 antibodies, researchers should:

• Test multiple fixation conditions (4% PFA, methanol, acetone) as epitope accessibility may be affected

• Optimize antigen retrieval methods (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

• Titrate antibody concentrations (typically starting at 1:100-1:500 dilutions)

• Include positive control tissues (based on Human Protein Atlas data showing RNF44 expression)

• Consider signal amplification systems for low-abundance expression

Note: Based on immunohistochemistry data, RNF44 shows variable expression across tissues, with higher levels observed in some cancer types, particularly HCC .

How should researchers design experiments to investigate the relationship between RNF44 expression and immune infiltration in cancer?

Based on correlative studies, RNF44 expression has been associated with various immune cell populations within the tumor microenvironment . To robustly investigate this relationship:

Recommended experimental design:

• Multiplex immunofluorescence to co-localize RNF44 with immune cell markers

• Single-cell RNA sequencing of tumor samples stratified by RNF44 expression levels

• Flow cytometric analysis of immune populations in RNF44-high vs. RNF44-low tumors

• Use conditional knockout models to assess causality between RNF44 and immune infiltration

Data from existing research indicates:

*P-values not explicitly stated in source material

What strategies can resolve contradictory findings regarding RNF44's role in disease pathogenesis?

When faced with conflicting data about RNF44's function:

Methodological approach to resolve contradictions:

• Perform meta-analysis of existing datasets with attention to tissue types, disease stages, and experimental methods

• Design targeted experiments with multiple model systems (cell lines, organoids, animal models)

• Analyze RNF44 isoform expression, as different splice variants may have distinct functions

• Employ CRISPR/Cas9-mediated knockout followed by rescue experiments with specific RNF44 mutants

• Consider post-translational modifications of RNF44 that might affect its function in different contexts

Studies have demonstrated that RNF44 overexpression correlates with poor prognosis in HCC , but the molecular mechanisms remain incompletely understood, necessitating deeper investigation.

How can researchers experimentally determine the specific ubiquitination targets of RNF44?

To identify and validate RNF44 ubiquitination substrates:

Recommended experimental workflow:

• Proximity-based labeling (BioID or APEX) with RNF44 as bait to identify interacting proteins

• Immunoprecipitation coupled with mass spectrometry under proteasome inhibition (MG132)

• Ubiquitin remnant profiling comparing wild-type vs. RNF44 knockout cells

• In vitro ubiquitination assays with recombinant RNF44 and candidate substrates

• Domain mapping to identify interaction regions between RNF44 and substrates

Note: Research on related RING finger proteins like RNF144B has shown specific ubiquitination of targets such as MDA5, suggesting potential parallels for studying RNF44 .

What approaches should be used to investigate RNF44's potential as a therapeutic target in cancer?

Based on its overexpression in HCC and correlation with poor prognosis , RNF44 presents a potential therapeutic target:

Recommended research strategy:

• Develop and screen small molecule inhibitors of RNF44's E3 ligase activity

• Employ the ARMeD (Antibody RING-Mediated Destruction) system to target RNF44 for degradation

• Design nanobody-based approaches similar to the DiffAb platform for targeted protein modulation

• Evaluate combination approaches with immune checkpoint inhibitors based on RNF44's correlation with immune infiltration

• Assess synthetic lethality approaches by identifying genes that, when inhibited together with RNF44, cause cancer cell death

How should researchers analyze the impact of RNF44 posttranslational modifications on its function and stability?

To investigate how PTMs affect RNF44:

Methodological approach:

• Phosphoproteomic analysis to identify phosphorylation sites on RNF44

• Site-directed mutagenesis of key residues to create phosphomimetic (S→D) or phospho-dead (S→A) mutants

• Analysis of RNF44 stability and localization using cycloheximide chase and cellular fractionation

• Investigation of auto-ubiquitination capacity of wild-type vs. mutant RNF44

• Identification of E2 conjugating enzymes that partner with RNF44 under different cellular conditions

Studies of related E3 ligases suggest that post-translational modifications can significantly alter substrate recognition, protein stability, and subcellular localization .

What factors affect the reliability of immunohistochemical staining with RNF44 antibodies?

When performing IHC with RNF44 antibodies, reliability can be affected by:

Critical technical factors:

• Antibody clone selection - polyclonal antibodies may give different staining patterns than monoclonals

• Epitope location - antibodies targeting different regions (N-terminal vs. internal epitopes) may yield varying results

• Fixation artifacts - overfixation may mask epitopes and lead to false-negative results

• Tissue processing variables - consistent sectioning and storage conditions are essential

• Interpretation criteria - clearly defined scoring systems for positive staining

To address these challenges, researchers should:

• Use Triple A Polyclonals that have been validated through the Human Protein Atlas project

• Include appropriate positive and negative control tissues

• Consider orthogonal validation methods (e.g., in situ hybridization)

• Blind scoring by multiple observers to reduce interpretation bias

How can researchers distinguish between acidic and basic species of RNF44 in complex samples?

Based on knowledge about antibody characterization for other proteins , RNF44 may exhibit charge variants that affect detection:

Methodological approach:

• Use ion exchange chromatography (IEX) to separate RNF44 charge variants

• Employ isoelectric focusing (IEF) followed by western blotting to resolve acidic and basic species

• Apply mass spectrometry to identify post-translational modifications that alter charge

• Perform 2D gel electrophoresis to separate RNF44 variants by both charge and molecular weight

• Use phosphatase or deglycosylation treatments to determine if PTMs contribute to charge heterogeneity

Researchers should be aware that modifications such as phosphorylation, deamidation, or oxidation may alter the electrophoretic mobility and antibody recognition of RNF44 .

What genetic knockdown approaches provide the most effective validation controls for RNF44 antibody specificity?

To generate reliable negative controls for antibody validation:

Recommended genetic approaches:

• Use commercially available RNF44 shRNA plasmids containing 3 different target-specific constructs to ensure complete knockdown

• Create CRISPR/Cas9 knockout cell lines targeting essential RNF44 domains

• Employ inducible shRNA systems to allow temporal control of RNF44 depletion

• Utilize siRNA pools targeting multiple regions of RNF44 mRNA

• Create epitope-specific knockout models if studying a particular region recognized by the antibody

Important considerations:

• Verify knockdown efficiency at both mRNA (qRT-PCR) and protein (western blot) levels

• Include appropriate transfection controls

• Consider potential compensation by related RING finger proteins

• Account for the half-life of the RNF44 protein when determining optimal timepoints for analysis after knockdown

How can researchers effectively design studies investigating RNF44's role in clinical outcomes of HCC patients?

Based on the correlation between RNF44 overexpression and poor prognosis in HCC :

Recommended study design:

• Prospective collection of matched tumor and adjacent normal tissues from HCC patients

• Comprehensive clinical data collection including age, weight, histologic grade, pathologic stage, AFP levels

• RNF44 expression analysis by IHC with standardized scoring system

• Stratification of patients into RNF44-high and RNF44-low groups based on median expression

• Multivariate Cox regression analysis controlling for clinical confounding factors

Current evidence demonstrates:

Source: Data extracted from

What methodologies should be employed to investigate the mechanistic relationship between RNF44 and cellular immune responses?

Given RNF44's correlation with immune infiltration and the known role of related RING finger proteins in immune regulation :

Recommended experimental approaches:

• Co-culture systems with RNF44-overexpressing tumor cells and various immune cell populations

• Transcriptome analysis of immune cells exposed to conditioned media from RNF44-manipulated cells

• ChIP-seq to identify potential transcriptional regulation of immune-related genes by factors downstream of RNF44

• Analysis of cytokine/chemokine profiles in RNF44-high vs. RNF44-low tumors

• Evaluation of immune checkpoint molecule expression in relation to RNF44 levels

Researchers should pay particular attention to the significant correlations observed between RNF44 expression and specific immune cell populations, especially T helper cells, Th2 cells, and cytotoxic cells .

How should researchers design experiments to investigate potential RNF44 variants and their functional impact?

To explore the functional diversity of RNF44 variants:

Methodological approach:

• RNA-seq analysis with junction-spanning reads to identify alternative splice variants

• PCR with variant-specific primers followed by sequencing validation

• Cloning and expression of identified variants to assess functional differences

• Domain-specific antibodies to detect truncated or alternatively spliced forms

• Bioinformatic prediction of functional consequences of identified variants