RNF181 Antibody

What is RNF181 and why is it important in cancer research?

RNF181, also known as HSPC238, is a 153 amino acid protein belonging to the RNF181 family that functions as an E3 ubiquitin-protein ligase. It regulates multiple cellular processes and is widely expressed throughout the body, with highest levels in liver and heart and lowest levels in brain and skeletal muscle . Recent research has established RNF181 as an important modulator of Hippo/YAP signaling in triple negative breast cancer (TNBC) progression. RNF181 stabilizes YAP protein by inhibiting K48-linked poly-ubiquitination, leading to enhanced YAP target gene expression and cancer progression . Data from multiple cohorts shows RNF181 is elevated in breast cancer compared to normal breast tissue, and correlates with poor survival in TNBC patients, suggesting its potential as a therapeutic target and prognostic marker .

What applications can the RNF181 antibody be used for?

The RNF181 antibody (such as catalog 20408-1-AP) has been validated for multiple research applications:

Each application requires specific optimization for successful RNF181 detection .

What cell lines and tissue samples show positive detection of RNF181?

Based on validation data, RNF181 antibody shows positive detection in:

For TNBC research specifically, BT549 and MDA-MB-231 cell lines have been used successfully to study RNF181 function .

What are the optimal dilutions for different applications of RNF181 antibody?

The following dilution ranges have been experimentally determined for optimal results:

Note that these are general guidelines, and each experimental system may require titration to obtain optimal signal-to-noise ratio .

How do I optimize Western blot protocols for RNF181 detection?

For optimal detection of RNF181 in Western blot applications:

• Sample preparation: Use RIPA buffer to harvest cell lysates, ensuring complete protein extraction .

• SDS-PAGE: RNF181 has an observed molecular weight of 18 kDa, so use gels with appropriate resolution in this range .

• Transfer: PVDF membranes are recommended for optimal protein retention and binding .

• Antibody dilution: Start with 1:1000 dilution in blocking buffer and adjust as needed for optimal signal .

• Detection: Use ECL system (such as GE RRPN 2235) for signal visualization .

• Controls: Include positive control samples known to express RNF181 (e.g., Jurkat or L02 cells) .

When investigating RNF181 interaction with YAP, be prepared to also detect YAP at approximately 65 kDa using appropriate YAP antibodies .

What antigen retrieval methods are effective for RNF181 IHC staining?

For successful immunohistochemical detection of RNF181:

The suggested antigen retrieval protocol is using TE buffer at pH 9.0. Alternatively, citrate buffer at pH 6.0 has also shown effectiveness . For optimal staining:

• Fix tissue samples appropriately (typically 4% paraformaldehyde).

• Perform antigen retrieval using TE buffer (pH 9.0) with heat treatment.

• Block with 3% BSA or appropriate blocking solution.

• Apply RNF181 antibody at 1:20-1:200 dilution (optimize for your specific tissue).

• Use appropriate detection system compatible with rabbit IgG primary antibodies.

Positive staining has been confirmed in human kidney and heart tissues, which can serve as positive controls .

How can I study protein-protein interactions involving RNF181?

To investigate RNF181 interactions with other proteins such as YAP:

• Immunoprecipitation (IP): Use 0.5-4.0 μg of RNF181 antibody per 1.0-3.0 mg of total protein lysate . Co-immunoprecipitation can identify protein binding partners.

• Co-localization studies:

  • Fix cells with 4% paraformaldehyde in PBS for 10 minutes

  • Permeabilize with 0.25% Triton X-100 for 5 minutes

  • Block with 3% BSA in PBS for 1 hour

  • Co-stain with anti-RNF181 antibody and antibodies against potential interacting proteins (e.g., anti-YAP)

  • Use appropriate secondary antibodies with different fluorescent conjugates

  • Analyze using confocal microscopy with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy Unit

  • Process images using software such as ImageJ

Research has confirmed RNF181 interaction with YAP through immunoprecipitation assays, showing that RNF181 promotes YAP stability by inhibiting K48-linked poly-ubiquitination .

How can I design RNF181 knockdown experiments to study its function?

For effective RNF181 knockdown experiments:

• siRNA approach: Use validated siRNA sequences targeting RNF181:

  • Option 1: CCACUGAUGACGACACUUAdTdT; UAAGUGUCGUCAUCAGUGGdTdT

  • Option 2: GAUGCCUUGCCAUCACCUUdTdT; AAGGUGAUGGCAAGGCAUCdTdT

• Transfection protocol:

  • Use RNAiMAX reagent (13778150, Invitrogen) for siRNA transfection

  • Transfect cells at 50-70% confluence

  • Include negative control siRNA (e.g., UUCUCCGAACGUGUCACGUTT; ACGUGACACGUUCGGAGAATT)

  • Validate knockdown efficiency by Western blot 48-72 hours post-transfection

• Rescue experiments:

  • Co-transfect siRNA-resistant RNF181 expression vector to confirm specificity

  • For functional studies, transfect YAP expression vectors to test if phenotypes can be rescued by YAP overexpression

Multiple siRNAs should be used to minimize off-target effects, as demonstrated in studies with BT549 and MDA-MB-231 cells .

What protocols are recommended for studying RNF181's effect on cell migration and invasion?

To assess RNF181's impact on cell migration and invasion in cancer cells:

• Wound healing assay:

  • Transfect cells with RNF181 siRNA or control siRNA

  • After 24 hours, seed cells in 6-well plates with 2% FBS

  • Once cells reach 100% confluence, create a straight scratch using a pipette tip

  • Measure wound distance at regular intervals (e.g., every 24 hours)

  • Calculate wound healing recovery as: [1 − (Width of the wound at a given time/width of the wound at t = 0)] × 100%

• Trans-well invasion assay:

  • Transfect cells with RNF181 siRNA or control siRNA

  • After 24 hours, seed cells in trans-well chambers with Matrigel-coated membranes

  • Add 20% serum to the lower chamber as a chemoattractant

  • Incubate for 16 hours

  • Remove non-invading cells

  • Fix and stain invaded cells with Crystal Violet

  • Count cell numbers using microscopy

These assays have revealed that RNF181 depletion significantly inhibits migration and invasion in TNBC cell lines, which can be rescued by YAP overexpression .

How does RNF181 regulate YAP protein stability and Hippo signaling?

To investigate RNF181's role in regulating YAP stability and Hippo signaling:

• Protein stability assays:

  • Transfect cells with siRNF181 or siControl

  • After 24 hours, treat cells with cycloheximide (100 μM)

  • Harvest cells at different time points (0, 2, 4, 8 hours)

  • Analyze YAP protein levels by Western blot

  • Calculate protein half-life by quantifying band intensity

• Ubiquitination assays:

  • Transfect cells with HA-tagged ubiquitin constructs (particularly K48-linked ubiquitin)

  • Co-transfect with Flag-RNF181 and/or Myc-YAP plasmids

  • Perform immunoprecipitation with YAP antibodies

  • Analyze ubiquitination by Western blot using anti-HA antibodies

  • Compare ubiquitination patterns with and without RNF181 expression

• Hippo signaling activity:

  • Measure TEAD luciferase activity using reporter assays

  • Assess Hippo/YAP target gene expression (CTGF, CYR61) by qPCR

  • Example primers and protocols can be found in the literature

Research has demonstrated that RNF181 stabilizes YAP by inhibiting its K48-linked poly-ubiquitination, leading to enhanced Hippo target gene expression and promoting cancer progression in TNBC .

How can I investigate the prognostic value of RNF181 in cancer patients?

To assess RNF181's prognostic significance:

• Database analysis:

  • Utilize public databases like Oncomine (https://www.oncomine.org) to compare RNF181 expression between cancer and normal tissues

  • Use KMPLOT database (http://kmplot.com/analysis/) to analyze correlation between RNF181 expression and patient survival

  • Analyze both 5-year and 10-year survival data for comprehensive assessment

• Tissue analysis:

  • Perform IHC on tissue microarrays containing cancer and adjacent normal tissues

  • Use RNF181 antibody at 1:20-1:200 dilution

  • Score staining intensity (e.g., 0-3 scale)

  • Correlate expression with clinicopathological parameters and patient outcomes

• Combined biomarker analysis:

  • Evaluate co-expression of RNF181 and YAP

  • Analyze correlation between RNF181/YAP expression and Hippo target genes

  • Investigate potential synergistic prognostic value

Research has shown that RNF181 is elevated in breast cancer compared to normal tissue in multiple cohorts, and correlates with poor survival in both general breast cancer patients and specifically in TNBC patients. Similar trends have been observed with YAP expression in TNBC patients .