rnf145 Antibody

What is RNF145 and what is its primary cellular function?

RNF145 is an ER-resident E3 ubiquitin ligase containing 14 transmembrane domains and a C-terminal RING domain essential for its ubiquitin ligase activity. It plays crucial roles in cholesterol biosynthesis regulation through sterol-dependent degradation of key regulatory proteins.

RNF145 mediates the degradation of HMG-CoA reductase (HMGCR), the rate-limiting enzyme in cholesterol biosynthesis, in response to high cellular sterol concentrations. Additionally, it triggers ubiquitination of SCAP (SREBP cleavage-activating protein) on lysine residues within cytoplasmic loops essential for COPII binding, inhibiting SCAP transport to the Golgi and subsequent processing of SREBP-2, a transcription factor that regulates cholesterol biosynthetic genes .

How is RNF145 expression regulated in cells?

RNF145 exhibits distinctive regulation patterns:

• Transcriptional regulation: RNF145 expression is induced by Liver X Receptor (LXR) activation. Treatment with LXR ligands such as GW3965 significantly increases RNF145 mRNA levels in hepatocytes, and this effect is abolished in LXR double-knockout mice .

• Sterol-responsive regulation: High-cholesterol diet feeding induces hepatic RNF145 expression in wild-type mice but not in LXRα/β knockout mice, indicating sterol-responsive, LXR-dependent regulation .

• Post-translational regulation: Endogenous RNF145 has a remarkably short half-life (~2 hours) and undergoes rapid proteasome-mediated degradation, contrasting with the stability of other E3 ligases like gp78. RNF145 appears to be auto-regulated through its own E3 ligase activity, as catalytically inactive RING domain mutants show greater stability .

What are the known protein interactions of RNF145?

RNF145 interacts with several key proteins in the cholesterol regulation pathway:

• Insig proteins: RNF145 contains a YLYF tetrapeptide motif similar to the YIYF motif in HMGCR and SCAP that mediates binding to Insig proteins. Endogenous RNF145 interacts with Insig-1 in a sterol-dependent manner, similar to SCAP and HMGCR interactions with Insigs .

• HMGCR: Under sterol-replete conditions, RNF145 is recruited to HMGCR via Insigs, leading to HMGCR ubiquitination and degradation. This interaction is lost in the absence of Insigs, indicating an absolute Insig-dependence .

• SCAP: RNF145 targets SCAP for ubiquitination, particularly on lysine residues K454 and K466 within a cytoplasmic loop that is essential for COPII binding and subsequent ER-to-Golgi transport .

• ADIPOR2: Beyond cholesterol metabolism, RNF145 has been shown to interact with ADIPOR2 (Adiponectin Receptor 2), mediating its ubiquitination and initiating its entry into the ER-associated degradation (ERAD) pathway .

What applications are RNF145 antibodies validated for?

According to the search results, commercially available RNF145 antibodies have been validated for multiple applications:

When selecting an antibody, researchers should verify that it has been validated for their specific application and target species, as reactivity varies between antibodies .

How can I optimize Western blot protocols for detecting endogenous RNF145?

Detecting endogenous RNF145 can be challenging due to its low basal expression levels and rapid turnover. Consider these optimization strategies:

• Sample preparation: Use UBE2G2 knockout cells or UBE2G2 inhibition to increase endogenous RNF145 levels, as RNF145 stability increases in the absence of its cognate E2 enzyme .

• Antibody dilution: Optimal dilution ranges for Western blotting are typically 1:500-1:1000 for commercially available antibodies .

• Molecular weight confirmation: Expect to observe RNF145 at 76-79 kDa on Western blots .

• Positive controls: Use HeLa cells, mouse kidney tissue, or HepG2 cells as positive controls, as these have been confirmed to express detectable levels of RNF145 .

• Sterol depletion: Consider treating cells with sterol depletion media to induce RNF145 expression before preparing lysates, as RNF145 mRNA expression increases approximately 3-fold under sterol depletion conditions .

What are the best experimental conditions for detecting RNF145-protein interactions?

To effectively detect interactions between RNF145 and its binding partners:

• Sterol manipulation: Most interactions with RNF145 are sterol-dependent. Use sterol depletion media followed by readdition of sterols (typically methyl-β-cyclodextrin-complexed cholesterol) to observe dynamic interactions .

• Stabilizing RNF145: Due to rapid turnover of endogenous RNF145, consider using a catalytically inactive mutant (C552A, H554A or C537A) which shows greater stability for interaction studies .

• Co-immunoprecipitation conditions: When performing co-IP experiments, UBE2G2 knockout cells can be used to increase endogenous RNF145 levels, making interactions easier to detect .

• Critical controls: Include the following controls:

  • HMGCR knockout cells when studying RNF145-Insig interactions to confirm HMGCR independence

  • Insig-1+2 knockout cells when studying RNF145-HMGCR interactions to confirm Insig dependence

  • Appropriate IgG controls for non-specific binding

How can I study the functional impact of RNF145 on cholesterol homeostasis in vivo?

Several methodological approaches have been validated for studying RNF145 function in vivo:

• Adenoviral-mediated overexpression: Transduction of C57BL/6 mice with adenoviruses encoding RNF145 has been shown to decrease serum cholesterol levels and suppress cholesterologenic gene expression within 6 days post-transduction .

• shRNA-mediated knockdown: Partial (40%) knockdown of endogenous RNF145 in mice using adenovirus-delivered shRNA results in modest increases in liver cholesterol content and upregulation of cholesterologenic genes .

• CRISPR/Cas9-mediated knockout: Complete genetic ablation of RNF145 in mice increases serum cholesterol levels in both LDL and HDL fractions compared to wild-type littermates .

• Genetic background considerations: Experiments in LDLR-knockout mice have demonstrated that RNF145's cholesterol-lowering effects are independent of LDLR-mediated lipoprotein clearance, suggesting direct effects on hepatic cholesterol biosynthesis .

For comprehensive analysis, researchers should consider measuring:

• Serum cholesterol levels (total and lipoprotein fractions)

• Liver cholesterol content

• Expression of cholesterologenic genes

• Nuclear abundance of mature SREBP-2

How does RNF145 cooperate with other E3 ligases in HMGCR degradation?

RNF145 functions within a complex network of E3 ligases that coordinate HMGCR degradation:

• Cooperative degradation with gp78: RNF145 and gp78 appear to independently coordinate HMGCR ubiquitination and degradation. While knockout of either RNF145 or gp78 alone has minimal effects on HMGCR degradation, simultaneous knockout of both ligases significantly impairs sterol-accelerated HMGCR degradation .

• Redundancy with Hrd1: In the absence of both RNF145 and gp78, the E3 ligase Hrd1 can partially regulate HMGCR activity. This suggests a hierarchical system of E3 ligases with built-in redundancy to ensure robust control of cholesterol biosynthesis .

• Experimental approach to study cooperation:

  • Generate single and double knockout cell lines using CRISPR/Cas9

  • Compare HMGCR degradation kinetics using cycloheximide chase assays

  • Quantify the relative contribution of each ligase using sterol-dependent HMGCR ubiquitination assays

  • Use UBE2G2 knockout cells to study the dependence of these ligases on specific E2 enzymes

What are the molecular mechanisms by which RNF145 regulates SREBP processing?

RNF145's regulation of SREBP processing involves specific molecular mechanisms:

• Ubiquitination of SCAP: RNF145 triggers ubiquitination of SCAP specifically on lysine residues K454 and K466 within cytoplasmic loop 6, which is essential for COPII binding and ER-to-Golgi transport .

• Inhibition of SREBP-2 processing: Expression of wild-type RNF145, but not catalytically inactive mutants or other ER-resident E3 ligases (GP78, TRC8), substantially inhibits sterol depletion-induced processing of SREBP-2 .

• Differential regulation of SREBP-1 vs SREBP-2 targets: While RNF145 initially suppresses both SREBP-1 and SREBP-2 target genes, SREBP-1 targets recover over time while SREBP-2 targets remain suppressed, suggesting complex regulatory mechanisms .

• Experimental approach to study this mechanism:

  • Use SCAP-knockout cells reconstituted with either wild-type SCAP or lysine-mutant SCAP (K454R, K466R)

  • Assess SREBP-2 processing under sterol depletion conditions with or without RNF145 expression

  • Measure nuclear abundance of mature SREBP-2 and expression of target genes

  • Monitor SCAP ubiquitination status using ubiquitination assays

How can I generate and validate RNF145 mutants for functional studies?

Creating functional RNF145 mutants is crucial for mechanistic studies:

• Key mutations for functional analysis:

  • RING domain mutations (C552A, H554A or C537A): Disrupts E3 ligase activity

  • YLYF tetrapeptide to AAAA mutations: Prevents Insig binding

  • Combined mutations: Can create double mutants to study both functions simultaneously

• Validation approaches:

  • In vitro ubiquitination assays with recombinant cytosolic domain (aa 511-663) to confirm loss of E3 ligase activity

  • Co-immunoprecipitation with Insigs to verify disruption of protein interactions

  • Functional rescue experiments in knockout cells to confirm biological activity

  • Western blotting to verify expression and stability of mutant proteins

• Expression systems:

  • Transient transfection for rapid analysis

  • Stable expression using lentiviral vectors (e.g., pHRSIN-P SFFV-P PGK-Hygromycin R) for long-term studies

  • Adenoviral delivery for in vivo studies

What techniques are most effective for studying sterol-dependent protein interactions with RNF145?

To effectively study the sterol-dependent interactions of RNF145:

• Sterol manipulation protocols:

  • Sterol depletion media: typically DMEM with 5% lipoprotein-deficient serum, 10 μM compactin, and 50 μM mevalonate for 16-24 hours

  • Sterol repletion: addition of 25-hydroxycholesterol (1 μg/ml) and cholesterol (10 μg/ml) for 1-4 hours

• Co-immunoprecipitation approaches:

  • For endogenous RNF145: Use UBE2G2 knockout cells to increase RNF145 stability

  • For overexpressed RNF145: Use epitope-tagged constructs (V5, FLAG) that don't interfere with protein interactions

  • Crosslinking may help capture transient interactions

• Proximity labeling techniques:

  • BioID or TurboID fusion proteins can identify neighboring proteins in intact cells

  • APEX2-based proximity labeling for rapid capturing of interacting proteins

  • These approaches are particularly valuable for membrane proteins like RNF145

• Mass spectrometry validation:

  • Immunoprecipitate epitope-tagged proteins and confirm interactions by mass spectrometry

  • Use quantitative proteomics approaches to measure sterol-dependent changes in protein interactions

What are the considerations for using RNF145 antibodies in immunohistochemistry studies?

For optimal immunohistochemistry (IHC) results with RNF145 antibodies:

• Antigen retrieval methods:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

  • Optimization may be required for specific tissue types

• Antibody dilution range:

  • Recommended dilution for IHC: 1:50-1:500

  • Sample-dependent optimization is essential

• Validated tissue samples:

  • Human lung cancer tissue has been validated for positive detection

  • Consider including known positive tissues as controls in each experiment

• Signal interpretation considerations:

  • RNF145 is an ER-resident protein, so staining should show characteristic ER pattern

  • Expression levels vary based on cellular sterol status

  • Sterol depletion or LXR agonist treatment may increase signal intensity

Why is my RNF145 signal weak or inconsistent in Western blot analyses?

Several factors may contribute to weak or inconsistent RNF145 detection:

• Low endogenous expression levels: RNF145 has naturally low basal expression. Consider:

  • Using LXR agonists (e.g., GW3965) to induce expression

  • Sterol depletion to upregulate RNF145 transcription

  • UBE2G2 knockout or inhibition to increase protein stability

• Rapid protein turnover: Endogenous RNF145 has a short half-life (~2 hours) and undergoes rapid proteasomal degradation. Consider:

  • Including proteasome inhibitors (MG132) in lysis buffers

  • Performing experiments in UBE2G2-deficient backgrounds

  • Using catalytically inactive RNF145 mutants for stronger signals

• Technical optimizations:

  • Transfer conditions: Optimize transfer time for high molecular weight proteins (~76-79 kDa)

  • Blocking agents: Test alternative blocking solutions (milk vs. BSA)

  • Signal enhancement: Consider using high-sensitivity ECL substrates

  • Antibody incubation: Extend primary antibody incubation to overnight at 4°C

How can I differentiate between the functions of RNF145 and other ERAD E3 ligases?

To distinguish RNF145's functions from other ERAD E3 ligases:

• Generate specific knockout models:

  • Single knockout lines (RNF145-KO, gp78-KO, Hrd1-KO)

  • Double knockout lines (RNF145/gp78-DKO)

  • Triple knockout models when necessary

  • Use CRISPR/Cas9 for clean genetic ablation

• Substrate specificity analysis:

  • Compare degradation kinetics of different substrates (HMGCR, SCAP, ADIPOR2)

  • Use domain swapping or chimeric proteins to identify specificity determinants

  • Perform global ubiquitinome analysis in different knockout backgrounds

• Regulation differences:

  • RNF145 is uniquely sterol-responsive at the transcriptional level

  • RNF145 has rapid turnover compared to more stable E3 ligases like gp78

  • LXR activation specifically induces RNF145 but not other ERAD E3 ligases

• Interaction partners:

  • RNF145 shows sterol-dependent interaction with Insig proteins

  • Different E3 ligases may preferentially interact with specific E2 enzymes

  • Co-immunoprecipitation followed by mass spectrometry can identify unique interaction partners

What controls should I include when studying sterol-dependent RNF145 function?

Critical controls for studying sterol-dependent RNF145 function include:

• Sterol manipulation controls:

  • Include both sterol-depleted and sterol-replete conditions

  • Use 25-hydroxycholesterol and cholesterol independently to distinguish oxysterol vs. cholesterol effects

  • Include LDLR-deficient models to rule out effects from lipoprotein uptake

• Protein interaction controls:

  • Include catalytically inactive RNF145 mutants (C552A, H554A) to distinguish between binding and ubiquitination effects

  • Use YLYF→AAAA mutants to confirm Insig-dependent interactions

  • HMGCR-knockout cells can confirm Insig-RNF145 interactions are HMGCR-independent

  • Insig-1/2 double knockout cells confirm HMGCR-RNF145 interactions require Insigs

• Transcriptional regulation controls:

  • Include LXRα/β double knockout models when studying sterol-dependent transcriptional regulation

  • Use cycloheximide to distinguish direct transcriptional effects from secondary effects requiring protein synthesis

  • Include time course analyses to capture dynamic regulation

• Methodological controls:

  • Perform ubiquitination assays with K48R or K63R ubiquitin mutants to determine ubiquitin chain specificity

  • Include proteasome inhibitors to confirm degradation mechanism

  • Use multiple independent antibodies to verify protein detection specificity