rnf144ab Antibody

What is RNF144B and why is it important in research?

RNF144B is an E3 ubiquitin ligase that plays significant roles in cellular processes including innate immune responses and apoptosis. Research has demonstrated that RNF144B interacts with the scaffold/dimerization domain (SDD) of TANK binding kinase 1 (TBK1) through its in-between RING (IBR) domain, inhibiting TBK1 phosphorylation and K63-linked polyubiquitination. This interaction ultimately leads to reduced IRF3 activation and decreased IFN-β production . RNF144B is also induced following DNA damage and has been implicated in cell death regulation. These diverse functions make RNF144B antibodies important tools for investigating immune regulation, cancer biology, and cellular stress responses.

What types of RNF144B antibodies are available for research applications?

Currently available RNF144B antibodies primarily include:

Most commercially available antibodies are rabbit polyclonal antibodies that recognize different epitopes of human RNF144B. Some antibodies also cross-react with mouse RNF144B, making them suitable for comparative studies across these species .

What are the common applications for RNF144B antibodies in research?

RNF144B antibodies are primarily used in:

• Western blotting (WB) - For detecting RNF144B protein expression levels and post-translational modifications

• Immunofluorescence/Immunocytochemistry (IF/ICC) - For visualizing subcellular localization, particularly membrane association

• Co-immunoprecipitation (Co-IP) - For studying protein-protein interactions, especially with TBK1

• Immunohistochemistry (IHC) - For examining tissue expression patterns

These applications allow researchers to investigate RNF144B's functional roles in various cellular contexts, particularly in inflammatory responses and cell death pathways .

How should I optimize Western blotting conditions for RNF144B detection?

When optimizing Western blotting for RNF144B detection:

• Sample preparation: Use RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS) supplemented with protease inhibitors .

• Gel separation: Use 10% or 4-15% gradient SDS-PAGE for optimal resolution, as RNF144B has a molecular weight of approximately 33-34 kDa .

• Antibody dilution: Start with dilutions between 1:500-1:3000 for primary antibody incubation . The optimal dilution should be determined experimentally.

• Detection system: Enhanced chemiluminescence (ECL) systems are generally suitable for RNF144B detection.

• Controls: Include positive controls (cells known to express RNF144B, such as heart, ovary, or testis tissue lysates) and negative controls (RNF144B knockdown samples) .

For membrane-bound RNF144B, cellular fractionation may be necessary to effectively detect the protein, as it contains a transmembrane domain that localizes it to cellular membranes .

What are the best approaches for studying RNF144B-TBK1 interactions?

To effectively study RNF144B-TBK1 interactions:

• Co-immunoprecipitation (Co-IP):

  • Harvest cells in TNN buffer (50 mM Tris, 0.25 M NaCl, 5 mM EDTA, 0.5% Nonidet P-40) supplemented with protease inhibitors

  • Perform immunoprecipitation using anti-RNF144B antibodies or epitope tag antibodies for tagged constructs

  • Detect TBK1 in the immunoprecipitates by Western blotting

• Proximity ligation assay (PLA):

  • This technique can visualize the interaction between RNF144B and TBK1 in situ

  • Use specific primary antibodies against RNF144B and TBK1 from different species

  • Follow with species-specific PLA probes and signal amplification

• Domain mapping:

  • Create constructs expressing specific domains of RNF144B, particularly focusing on the IBR domain

  • Perform pulldown assays with the SDD domain of TBK1

  • This approach helps identify the critical interaction regions

When studying these interactions, stimulation with LPS at different time points (0, 15, 30, 60, 120 min) is recommended to observe dynamic changes in the interaction .

How can I effectively validate RNF144B antibody specificity?

To validate RNF144B antibody specificity:

• siRNA knockdown validation:

  • Transfect cells with RNF144B-specific siRNAs

  • Compare protein detection in knockdown vs. control cells by Western blot

  • A specific antibody will show decreased signal intensity in knockdown samples

• Overexpression controls:

  • Transfect cells with RNF144B expression vectors

  • Compare detection in overexpression vs. empty vector controls

  • A specific antibody will show increased signal in overexpressing cells

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide

  • Compare detection with and without peptide competition

  • A specific antibody will show signal reduction after peptide competition

• Cross-reactivity testing:

  • Test the antibody against related proteins (e.g., RNF144A) to ensure specificity

  • This is particularly important since RNF144B shares structural similarities with RNF144A

• Multiple antibody validation:

  • Use multiple antibodies targeting different epitopes

  • Consistent detection patterns across different antibodies support specificity

How can I investigate RNF144B's role in LPS-induced inflammatory responses?

To investigate RNF144B's role in LPS-induced inflammatory responses:

• Expression profiling:

  • Treat cells (THP-1 human monocytic cells or human monocyte-derived macrophages) with LPS (typically 100 ng/ml)

  • Monitor RNF144B expression at different time points (0-24h) using qRT-PCR and Western blot

  • Compare with inflammatory gene expression (e.g., IFN-β, TNF-α)

• Knockdown/overexpression studies:

  • Use siRNA to knockdown RNF144B (validated sequences from published studies)

  • Alternatively, overexpress RNF144B using expression vectors

  • Stimulate with LPS and measure effects on:

    • TBK1 phosphorylation

    • IRF3 phosphorylation and nuclear translocation

    • IFN-β production (ELISA or qRT-PCR)

• Mechanism investigation:

  • Perform co-IP to assess RNF144B-TBK1 interaction dynamics after LPS stimulation

  • Examine TBK1 K63-linked polyubiquitination in the presence/absence of RNF144B

  • Use luciferase reporter assays with IFN-β promoter constructs to measure transcriptional activation

• Signaling pathway analysis:

  • Test the effect of MyD88 inhibition or NF-κB inhibition (using BAY-11-7085) on RNF144B induction

  • Evaluate responses to different TLR ligands (LTA, poly(I:C), flagellin, R848, CpG ODNs)

This approach provides a comprehensive understanding of how RNF144B functions as a negative regulator of LPS-induced inflammation.

What are the key considerations when comparing RNF144B with the related protein RNF144A?

When comparing RNF144B with RNF144A:

• Structural comparisons:

  • Both contain RING-IBR-RING (RBR) domains and a transmembrane domain

  • RNF144A has a transmembrane domain with a G₂₅₂XXG₂₅₆ motif important for self-association

  • RNF144B likely has similar structural features but may have distinct functional domains

• Localization studies:

  • Use immunofluorescence with specific antibodies to compare subcellular localization

  • Employ subcellular fractionation to quantitatively assess membrane vs. cytosolic distribution

  • Create chimeric constructs to identify determinants of differential localization

• Functional differences:

  • RNF144A targets DNA-PKcs for ubiquitination and degradation

  • RNF144B interacts with TBK1 to inhibit its activity without degradation

  • Compare effects on cell death pathways and inflammatory responses

• Self-association properties:

  • Both proteins demonstrate self-association mediated by their transmembrane domains

  • Use co-IP and cross-linking assays to compare oligomerization properties

  • Evaluate the impact of transmembrane domain mutations on function

• E3 ligase activity:

  • Compare substrate specificity using in vitro and in vivo ubiquitination assays

  • Assess the importance of the RBR domains in both proteins for their respective functions

Understanding these similarities and differences provides insight into the specialized functions of these related E3 ubiquitin ligases.

How can I design experiments to investigate post-translational modifications of RNF144B?

To investigate post-translational modifications of RNF144B:

• Phosphorylation analysis:

  • Immunoprecipitate RNF144B from cell lysates using specific antibodies

  • Perform Western blotting with phospho-specific antibodies (if available)

  • Alternatively, use Phos-tag SDS-PAGE to separate phosphorylated forms

  • Mass spectrometry analysis of immunoprecipitated RNF144B can identify specific phosphorylation sites

• Ubiquitination studies:

  • Transfect cells with HA-tagged ubiquitin and FLAG-tagged RNF144B

  • Treat with proteasome inhibitors (e.g., MG132, 20 μM for 6-8 hours)

  • Immunoprecipitate RNF144B under denaturing conditions (1% SDS, boiling)

  • Detect ubiquitination by Western blotting with anti-HA antibodies

• SUMOylation and neddylation analysis:

  • Co-express RNF144B with tagged SUMO or NEDD8

  • Immunoprecipitate under denaturing conditions

  • Detect modifications by Western blotting

• Mutation studies:

  • Identify potential modification sites using bioinformatics

  • Create site-specific mutants (e.g., lysine to arginine for ubiquitination sites)

  • Compare functional consequences of mutations on:

    • Protein stability

    • Subcellular localization

    • Interaction with binding partners (e.g., TBK1)

    • E3 ligase activity

• Stimulus-dependent modification:

  • Examine how inflammatory stimuli (LPS) or cell stress affect RNF144B modifications

  • Use time-course experiments to capture dynamic changes

These approaches will provide insight into how post-translational modifications regulate RNF144B function in different cellular contexts.

Why might I observe inconsistent RNF144B detection in Western blot experiments?

Inconsistent RNF144B detection in Western blots may result from:

• Expression dynamics:

  • RNF144B expression is inducible by stimuli like LPS in a time-dependent manner

  • Expression peaks approximately 6 hours after LPS stimulation in human monocytic cells

  • Inconsistent timing of sample collection may result in variable detection

• Membrane localization:

  • RNF144B contains a transmembrane domain that anchors it to cellular membranes

  • Inefficient extraction from membranes using standard lysis buffers can cause inconsistent detection

  • Solution: Use lysis buffers containing stronger detergents or perform subcellular fractionation

• Antibody specificity:

  • Some antibodies may cross-react with related proteins (e.g., RNF144A)

  • Different antibodies may recognize distinct epitopes with varying accessibility

  • Solution: Validate antibodies using knockdown controls and test multiple antibodies

• Post-translational modifications:

  • Modifications may mask epitopes or alter migration patterns

  • Solution: Use denaturing conditions and consider the impact of treatments on protein modifications

• Protein stability:

  • RNF144B may be subject to rapid turnover under certain conditions

  • Solution: Consider using proteasome inhibitors (e.g., MG132) when appropriate

To improve consistency, standardize sample collection timing, optimize extraction protocols for membrane proteins, and validate antibody specificity thoroughly.

What are the critical factors for successful immunoprecipitation of RNF144B?

For successful immunoprecipitation of RNF144B:

• Lysis buffer optimization:

  • Use TNN buffer (50 mM Tris, 0.25 M NaCl, 5 mM EDTA, 0.5% Nonidet P-40) supplemented with:

    • 1 mM dithiothreitol

    • 1 mM NaF

    • 1 mM sodium orthovanadate

    • 20 nM microcystin

    • Protease inhibitor cocktail

  • This buffer effectively solubilizes membrane-associated proteins while preserving interactions

• Epitope tag considerations:

  • When possible, use epitope-tagged constructs (FLAG, HA, Myc) for more efficient immunoprecipitation

  • Position tags carefully to avoid interfering with the transmembrane domain or functional domains

• Antibody selection:

  • Test multiple antibodies for immunoprecipitation efficiency

  • Some antibodies work well for Western blot but poorly for immunoprecipitation

  • Consider using protein A/G beads pre-coupled with antibodies for more efficient capture

• Cross-linking (for protein complexes):

  • For detecting self-association or weak interactions, consider using chemical cross-linkers

  • Dimethyl pimelimidate-2HCl (20 mM) has been successfully used for RNF144B

• Controls:

  • Include IgG control immunoprecipitations

  • Use knockdown or knockout cells as negative controls

  • For overexpressed constructs, compare with empty vector controls

• Denaturing conditions (for studying modifications):

  • For ubiquitination studies, use denaturing conditions (1% SDS lysis buffer, boiling)

  • Dilute samples 1:10 in TNN buffer before immunoprecipitation

Following these guidelines will improve the specificity and efficiency of RNF144B immunoprecipitation experiments.

How can I resolve difficulties in detecting endogenous RNF144B in different cell types?

To improve detection of endogenous RNF144B across different cell types:

• Cell type selection:

  • RNF144B is broadly expressed but with variable levels across tissues

  • Higher expression is reported in heart, ovary, and testis tissues

  • Lower expression in brain and thymus

  • In immune cells, expression is inducible by LPS and other TLR ligands

• Stimulation protocols:

  • For immune cells (THP-1, HMDM), stimulate with LPS (100 ng/ml) for 6 hours

  • For other cell types, consider DNA damage inducers as RNF144B is p73-inducible upon DNA damage

• Enrichment strategies:

  • Perform subcellular fractionation to enrich membrane-associated RNF144B

  • Immunoprecipitate RNF144B before Western blotting to concentrate the protein

  • Use MACS or FACS to isolate specific cell populations from tissues

• Signal amplification:

  • Employ more sensitive detection methods such as chemiluminescence or fluorescence

  • Consider using biotin-streptavidin amplification systems

  • For immunofluorescence, use signal amplification methods like tyramide signal amplification

• Antibody combinations:

  • Use a cocktail of validated RNF144B antibodies targeting different epitopes

  • This approach can increase sensitivity while maintaining specificity

• Preparation of concentrated lysates:

  • Use larger amounts of starting material

  • Concentrate proteins using TCA precipitation or similar methods before analysis

These approaches should help overcome detection challenges for endogenous RNF144B across different experimental systems.

What experimental approaches can I use to study RNF144B E3 ligase activity?

To study RNF144B E3 ligase activity:

• In vitro ubiquitination assays:

  • Purify GST-tagged RNF144B or immunoprecipitate it from cells

  • Combine with purified E1, E2 (typically UbcH7), and HA-tagged ubiquitin

  • Include ATP and an ATP regeneration system in the reaction buffer

  • Detect ubiquitination by Western blotting with anti-HA antibodies

  • Controls should include reactions lacking E1, E2, or ATP

• In vivo ubiquitination assays:

  • Co-transfect cells with HA-tagged ubiquitin and FLAG-tagged RNF144B

  • Treat with proteasome inhibitor MG132 (20 μM, 6-8 hours)

  • Lyse cells in denaturing buffer (1% SDS, 60 mM Tris pH 6.8)

  • Boil lysates, dilute in TNN buffer, immunoprecipitate substrate proteins

  • Detect ubiquitination by Western blotting with anti-HA antibodies

• Structure-function analysis:

  • Create domain mutants focusing on the RING and IBR domains

  • Common mutations include cysteine-to-alanine substitutions in the RING domains

  • Compare ubiquitination activity of wildtype vs. mutant proteins

• Substrate identification:

  • Perform immunoprecipitation-mass spectrometry to identify potential substrates

  • Confirm with directed ubiquitination assays on candidate substrates

  • Examine protein stability of substrates in the presence/absence of RNF144B

• E2 enzyme specificity:

  • Test multiple E2 enzymes to determine specificity

  • UbcH7 has been used successfully with related RBR E3 ligases

These approaches provide a comprehensive analysis of RNF144B's enzymatic activity and substrate specificity.

How can I design experiments to dissect the membrane localization requirements of RNF144B?

To dissect membrane localization requirements of RNF144B:

• Domain deletion and mutation analysis:

  • Create a transmembrane domain deletion mutant (ΔTM, similar to Δa.a. 250-270 for RNF144A)

  • Generate point mutations in the transmembrane domain, focusing on hydrophobic residues

  • A 3L→R mutation strategy (similar to 3L259R in RNF144A) can disrupt membrane insertion

• Localization imaging:

  • Express fluorescently tagged RNF144B constructs (wildtype and mutants)

  • Perform live-cell imaging to track localization

  • Use fixed-cell immunofluorescence with organelle markers for co-localization studies

  • Compare localization patterns before and after cellular stimulation

• Subcellular fractionation:

  • Separate cells into cytosolic, nuclear, and membrane fractions

  • Use established markers to validate fractionation (GAPDH for cytosol, p84 for nucleus, EGFR for membrane)

  • Analyze distribution of wildtype and mutant RNF144B by Western blotting

• Membrane insertion analysis:

  • Perform in vitro membrane insertion assays using purified microsomes

  • Test protease protection to determine topology

  • Use glycosylation mapping to identify lumenal domains

• Chimeric protein analysis:

  • Swap the transmembrane domain of RNF144B with those from other proteins

  • Determine whether membrane localization is sufficient for function

  • Identify specific sequences required for proper localization

These approaches will define the requirements for RNF144B membrane localization and its functional significance.

What methods are recommended for investigating RNF144B's role in regulating TBK1 activity?

To investigate RNF144B's role in regulating TBK1 activity:

• Phosphorylation analysis:

  • Manipulate RNF144B levels (knockdown or overexpression)

  • Stimulate cells with LPS at different time points (0, 15, 30, 60, 120 min)

  • Detect TBK1 phosphorylation (Ser172) by Western blotting

  • Quantify phosphorylation levels relative to total TBK1

• K63-linked ubiquitination analysis:

  • Immunoprecipitate TBK1 under denaturing conditions

  • Probe for K63-specific ubiquitin chains using K63-linkage specific antibodies

  • Compare ubiquitination levels with and without RNF144B expression

• Domain mapping of the interaction:

  • Create constructs expressing specific domains of RNF144B (focus on the IBR domain)

  • Test interaction with the scaffold/dimerization domain (SDD) of TBK1

  • Use co-IP assays to map critical interaction surfaces

• Downstream signaling analysis:

  • Monitor IRF3 phosphorylation and nuclear translocation

  • Measure IFN-β promoter activity using luciferase reporter assays

  • Quantify IFN-β mRNA and protein production by qRT-PCR and ELISA

• Structure-function analysis:

  • Generate point mutations in the IBR domain of RNF144B

  • Assess their effects on TBK1 binding and inhibition

  • Determine whether E3 ligase activity is required for TBK1 inhibition

• In vivo relevance:

  • Use mouse models with RNF144B knockout or knockdown

  • Challenge with LPS and measure inflammatory responses

  • Assess susceptibility to endotoxic shock or bacterial infections