RPL13 Antibody

What is RPL13 protein and what are its canonical functions?

RPL13 (ribosomal protein L13) is a component of the 60S large ribosomal subunit with a calculated and observed molecular weight of 24 kDa . It is encoded by the RPL13 gene (Gene ID: 6137) and functions primarily in ribosome assembly and protein translation . The mammalian ribosome comprises 79 ribosomal proteins and four rRNAs, which combine in equimolar ratios to form the small (40S) and large (60S) subunits . RPL13 plays a critical role in the formation of peptide bonds during protein synthesis. It is distinct from RPL13A, which has more extraribosomal functions and is not required for canonical ribosome function .

What applications is the RPL13 antibody validated for?

RPL13 antibody has been validated for multiple research applications as shown in the following table:

The antibody (e.g., 11271-1-AP) targets RPL13 in these applications and shows reactivity with human, mouse, and rat samples .

How should I optimize antigen retrieval for RPL13 immunohistochemistry?

For optimal immunohistochemical detection of RPL13, two antigen retrieval methods have shown effectiveness:

• Primary recommendation: Use TE buffer at pH 9.0 for antigen retrieval, which often yields superior results for nuclear proteins like RPL13 .

• Alternative method: Citrate buffer at pH 6.0 can also be used if the primary method doesn't yield satisfactory results .

The choice between these methods may depend on your specific tissue type and fixation protocol. It is advisable to run parallel experiments with both retrieval methods to determine which works best for your specific experimental conditions.

What extraribosomal functions does RPL13 perform in cellular processes?

RPL13 performs several critical extraribosomal functions beyond its role in protein synthesis:

• Immune response regulation: RPL13 interacts with retinoic acid-inducible gene I (RIG-I) and binds to the 3′ untranslated region (3′-UTR) of NF-κB1 mRNA to promote its translation, thereby enhancing NF-κB activation and downstream inflammatory gene expression .

• Antiviral activity: RPL13 participates in the antiviral immune response, particularly against foot-and-mouth disease virus (FMDV), by promoting the induction and activation of NF-κB and interferon-β (IFN-β) genes .

• Cytokine production: Overexpression of RPL13 enhances the expression and secretion of IFN-β and the proinflammatory cytokine interleukin-6 (IL-6), while knockdown of RPL13 has the opposite effects .

• Tumor suppression: Some studies suggest RPL13 may function as a tumor suppressor gene, although further research is needed to fully characterize this role .

How does RPL13 contribute to viral IRES-driven translation?

RPL13 serves as a critical regulator of internal ribosome entry site (IRES)-driven translation during FMDV infection. The mechanism involves:

• Direct interaction: RPL13 is identified as a key factor in facilitating IRES-driven translation of FMDV .

• Co-factor recruitment: Research supports a model whereby FMDV IRESs recruit helicase DDX3, which recognizes RPL13 to facilitate IRES-driven translation, with assistance from eIF3e and eIF3j subunits .

• Broader implications: This mechanism is not unique to FMDV but extends to other viruses like Seneca Valley virus (SVV) in the Picornaviridae family .

This specialized function of RPL13 is significant as it represents a selective translation mechanism for viral mRNAs, which could potentially be targeted for viral infection prevention strategies.

How does RPL13 participate in innate immune responses against viral infection?

RPL13 plays a multifaceted role in antiviral immunity:

• Activation of signaling pathways: RPL13 promotes the induction and activation of NF-κB and IFN-β gene promoters, enhancing antiviral responses .

• Antagonism by viral proteins: FMDV 3C protease (3Cpro) interacts with RPL13 and reduces its expression, thus antagonizing RPL13-mediated antiviral activity. This represents a viral evasion strategy to maintain replication .

• Comparative function: Unlike RPL13, the related protein RPL13A acts as a negative regulator of inflammatory responses. In the late stage of interferon-γ stimulation, RPL13A forms part of the IFN-γ-activated inhibitor of translation (GAIT) complex, which selectively inhibits translation of inflammatory genes to control excessive inflammation .

This dual role of ribosomal proteins in both promoting and regulating immune responses highlights their importance in balancing effective immunity while preventing inflammatory damage.

What genetic associations exist between RPL13 variants and human diseases?

Research has established important associations between RPL13 variants and skeletal disorders:

• Spondyloepimetaphyseal dysplasia (SEMD-RPL13 type): Monoallelic variants in RPL13 are associated with this skeletal dysplasia in multiple unrelated families .

• Novel findings on penetrance: Recent research has uncovered incomplete penetrance and broad phenotypic variability in SEMD-RPL13 type, even within families harboring identical mutations .

• Molecular mechanisms: Cellular defects in affected individuals include reduction in 80S ribosomes and attenuated global translation, confirming impaired ribosomal function .

• Specific mutations: Two novel missense RPL13 mutations (p.A178D and p.A185P) affecting the C-terminal α-helix of eL13 have been identified, as well as previously reported splicing variants .

This represents a novel ribosomopathy with major skeletal involvement but absent extra-skeletal manifestations, expanding our understanding of ribosomal protein mutations in human disease.

What controls should be included when using RPL13 antibody in experimental workflows?

For rigorous experimental design using RPL13 antibody, the following controls are essential:

• Positive controls: Use tissues or cell lines with confirmed RPL13 expression such as:

  • HeLa cells

  • SGC-7901 cells

  • Human lung tissue

  • Human colon cancer tissue

• Negative controls:

  • Isotype control: Include rabbit IgG at matching concentrations to confirm specificity

  • Knockdown validation: Use siRNA or shRNA against RPL13 to demonstrate antibody specificity

  • Blocking peptide: Pre-incubate antibody with immunogen peptide to demonstrate specific binding

• Loading controls: For western blots, use established housekeeping proteins appropriate for your experiment, considering that RPL13 itself is often used as a reference gene in some contexts.

How should RPL13 antibody be stored and handled to maintain optimal activity?

To preserve antibody functionality:

What approaches can be used to characterize RPL13 expression at the protein level?

For comprehensive protein-level characterization of RPL13:

• Western blotting protocol:

  • Grow cells to 90% confluence

  • Lyse in ice-cold RIPA buffer containing protease inhibitors

  • Collect lysate by centrifugation

  • Quantify total protein (e.g., using BCA assay)

  • Analyze 10μg of denatured protein samples

  • Use primary antibody against RPL13 (e.g., mouse monoclonal, 1:1000 dilution)

  • Detect with HRP-conjugated secondary antibody (e.g., anti-mouse, 1:20000 dilution)

• Immunoprecipitation approach:

  • Use 0.5-4.0μg of antibody for 1.0-3.0mg of total protein lysate

  • Validated in HeLa cells

• Subcellular localization studies:

  • Immunofluorescence staining using validated antibodies

  • Co-staining with nucleolar markers to confirm ribosomal association

How does RPL13 expression change during viral infection, and what mechanisms drive these changes?

During viral infection, RPL13 undergoes significant expression changes:

• Viral antagonism: FMDV 3C protease (3Cpro) directly interacts with RPL13 and reduces its expression to counteract RPL13-mediated antiviral activity .

• Mechanism of degradation: FMDV maintains its own replication by antagonizing RPL13-mediated resistance through the degradation of RPL13 .

• Regulatory shift: During respiratory syncytial virus (RSV) infection, the related protein RPL13A is released from the 60S large subunit and forms a virus-activated translation inhibition complex (VAIT) when it recognizes specific hairpin structures in viral mRNA 3′-UTR .

• Comparative viral strategies: Different viruses interact with ribosomal proteins in unique ways:

  • Rabies virus P protein interacts with RPL9, promoting its translocation from nucleus to cytoplasm to inhibit viral transcription

  • RPL19 participates in TLR3-receptor-mediated signaling pathways to inhibit vesicular stomatitis virus (VSV) replication

What considerations are important when using RPL13 as a reference gene in gene expression studies?

While RPL13 is sometimes used as a reference gene, researchers should consider:

• Context dependency: RPL13 may not be stably expressed under all experimental conditions, particularly during viral infections when its expression can be specifically targeted by viral proteins .

• Validation requirement: Before using RPL13 as a reference gene, perform stability analysis across your experimental conditions using algorithms like geNorm, NormFinder, or BestKeeper.

• Alternative approaches: Consider using multiple reference genes simultaneously for normalization to improve reliability.

• Specific contraindications: Avoid using RPL13 as a reference gene when:

  • Studying ribosomal biogenesis or function

  • Analyzing cellular responses to viral infection, particularly picornaviruses

  • Investigating skeletal dysplasias where RPL13 mutations may be present

• Extraribosomal functions: Be aware that RPL13's involvement in immune signaling and viral replication could confound interpretation if used as a reference gene in related studies.

How can RPL13 be utilized as a target for antiviral therapeutic development?

RPL13's role in viral replication offers several potential therapeutic avenues:

• Blocking viral protease-RPL13 interactions: Development of small molecule inhibitors that prevent FMDV 3Cpro from degrading RPL13 could restore antiviral activity .

• Enhancing RPL13-mediated immune responses: Compounds that potentiate RPL13's ability to activate NF-κB and IFN-β might strengthen innate immunity against viral infection .

• Targeting RPL13's role in IRES-driven translation: Disrupting the interaction between RPL13, DDX3, and viral IRES elements could inhibit viral protein synthesis without affecting cap-dependent host translation .

• Comparative strategies: Drawing from the understanding of how respiratory syncytial virus and rabies virus interact with ribosomal proteins could provide additional therapeutic targets .

The specificity of these interactions makes them attractive for developing antivirals with potentially fewer side effects than broad-spectrum approaches.

What techniques are emerging for studying RPL13 localization and dynamics during cellular stress?

Advanced techniques for studying RPL13 dynamics include:

• Live-cell imaging: Using tagged RPL13 constructs to monitor real-time changes in localization during stress responses or viral infection.

• Proximity labeling techniques: BioID or APEX2 fusions with RPL13 to identify stress-specific interaction partners.

• Ribosome profiling: To determine how RPL13 variants affect ribosome occupancy on specific mRNAs during stress conditions.

• CRISPR-Cas9 base editing: For introducing specific RPL13 variants to study structure-function relationships without complete gene disruption.

• Cryo-electron microscopy: To determine structural changes in ribosomes containing mutant forms of RPL13 associated with skeletal dysplasias.

These emerging approaches will help clarify how RPL13 transitions between its canonical ribosomal role and its extraribosomal functions during cellular stress and disease states.