PPP1R13L Antibody

What is PPP1R13L and why is it important in research?

PPP1R13L (Protein Phosphatase 1, Regulatory Subunit 13 Like), also known as iASPP, is a highly conserved inhibitor of p53 that selectively regulates a subset of p53 target genes. It was initially identified as a protein that binds to the NF-κB subunit p65/RelA and inhibits its transcriptional activity . PPP1R13L has emerged as an important research target due to its involvement in tumor progression, particularly in cervical cancer, acute myeloid leukemia, and other malignancies . It plays critical roles in cellular processes including apoptosis regulation, cell proliferation, epithelial-mesenchymal transition, and glycolysis, making it a significant target for cancer research .

What are the different isoforms of PPP1R13L and how do they affect antibody selection?

PPP1R13L exists in multiple isoforms, with isoform 1 (~100 kDa) and isoform 2 (~50 kDa) being the most commonly studied . When selecting an antibody, researchers should consider which isoform they intend to detect. Some antibodies are specifically designed to target epitopes present in isoform 1, such as those binding to AA 775-800 or AA 780-797 . Additionally, PPP1R13L can be cleaved by Caspase-3, producing a stable PPP1R13L (295-828aa) fragment that relocates from the cytoplasm to the nucleus, which is critical for its inhibition of p53 transcription . For studies focusing on the nuclear functions of PPP1R13L, antibodies recognizing the C-terminal region containing the Ank-SH3 domain would be most appropriate.

What are the synonyms for PPP1R13L that researchers should be aware of?

When searching literature or antibody databases, researchers should be aware of multiple synonyms for PPP1R13L, including:

• iASPP (inhibitor of apoptosis stimulating protein of p53)

• Inhibitor of ASPP protein

• RelA-associated inhibitor (RAI)

• NFkB interacting protein 1 (NKIP1)

• PPP1R13B-like protein (PPP1R13BL)

This awareness is crucial for comprehensive literature searches and avoiding confusion when comparing research findings that use different nomenclature.

What are the validated applications for PPP1R13L antibodies in cancer research?

PPP1R13L antibodies have been validated for multiple applications in cancer research:

Research indicates that PPP1R13L promotes cervical cancer progression by suppressing p63-mediated PTEN transcription, leading to activation of the AKT/mTOR pathway . In acute myeloid leukemia, high iASPP expression correlates with poor clinical outcomes, making antibody-based detection methods valuable prognostic tools .

How can PPP1R13L antibodies be used to study its role in apoptosis regulation?

PPP1R13L antibodies can be employed to investigate its dual role in apoptosis regulation through several methodological approaches:

• Co-immunoprecipitation experiments: Use PPP1R13L antibodies to pull down protein complexes and analyze interactions with p53, p63, p73, and NF-κB subunits to understand how PPP1R13L mediates apoptotic signaling .

• Chromatin immunoprecipitation (ChIP): Employ PPP1R13L antibodies in ChIP assays to examine how PPP1R13L affects p53 family member binding to promoters of pro-apoptotic genes like PTEN .

• Immunofluorescence: Detect subcellular localization changes of PPP1R13L following apoptotic stimuli, particularly its translocation from cytoplasm to nucleus after Caspase-3 cleavage .

• Western blotting: Measure PPP1R13L expression levels in cells undergoing apoptosis, potentially revealing correlation between PPP1R13L levels and apoptotic resistance in cancer cells .

Research has shown that PPP1R13L can have both pro-apoptotic and anti-apoptotic effects depending on the cellular context and experimental conditions , making careful antibody selection and experimental design crucial.

What are the recommended dilutions and conditions for using PPP1R13L antibodies in different applications?

For optimal results, researchers should always perform titration experiments to determine the ideal antibody concentration for their specific experimental system and sample type .

What cross-reactivity should be considered when selecting a PPP1R13L antibody?

When selecting a PPP1R13L antibody, consider these cross-reactivity factors:

• Species reactivity: Different antibodies show varying reactivity profiles. For example:

  • ABIN129682: Reactive with human samples only

  • ABIN401339: Reactive with human, mouse, and dog samples, with partial reactivity to bovine specimens

  • 51141-1-AP: Validated for human and mouse samples

• Isoform specificity: Ensure the antibody recognizes the specific isoform of interest. Some antibodies are designed to target specific amino acid regions like AA 775-800 or AA 780-797 in isoform 1 .

• Domain recognition: For functional studies, select antibodies that recognize specific domains such as the Ank-SH3 domain, which mediates interactions with p53 family members .

• Related protein discrimination: Verify the antibody can discriminate between PPP1R13L and its family members ASPP1 and ASPP2, which share structural similarity in their C-terminal regions .

Cross-reactivity information should be experimentally validated in your system, especially when working with less commonly studied species or tissue types.

How can antibodies help elucidate the selective regulation of p53 family target genes by PPP1R13L?

Understanding the sequence-specific regulation of p53 family target genes by PPP1R13L requires sophisticated antibody-based approaches:

• ChIP-seq analysis: Use PPP1R13L antibodies in ChIP-seq experiments to identify genome-wide binding sites. Research has identified sequence signatures of PPP1R13L-regulated p53 response elements (REs), which feature C9 and/or G12 in addition to the typical p53 RE characteristics .

• Re-ChIP experiments: Sequential ChIP using antibodies against PPP1R13L followed by p53, p63, or p73 antibodies to identify regions where these proteins co-localize on DNA.

• Proximity ligation assays: Visualize and quantify specific PPP1R13L-p53 family protein interactions at different p53 target gene promoters using paired antibodies.

• CRISPR-engineered mutant studies: Create specific mutations in the response elements of PPP1R13L-regulated genes (like the identified site1 in PTEN with pattern of C4, G7, C9, A12, C14, and G17), then use antibodies to assess altered binding patterns .

Research has shown that PPP1R13L specifically inhibits the transcriptional activity of TAp63 on PTEN through interaction with a specific response element, with C9 being a key element in this selective regulation . This discovery provides a sequence basis for understanding how PPP1R13L selectively regulates only certain p53 family target genes.

How does p53 mutation status affect PPP1R13L function and antibody-based detection methods?

The interaction between PPP1R13L and mutant p53 presents unique challenges for antibody-based research:

• Opposing functional effects: In C33A cells (HPV-negative cervical cancer cells with p53 R273C mutation), PPP1R13L exhibits effects opposite to those observed with wild-type p53. Specifically, in these cells, PPP1R13L paradoxically promotes rather than inhibits the transcriptional activity of certain promoters .

• Tetramer formation considerations: Since p53 functions as a tetramer, even overexpression of wild-type p53 cannot reverse the effects of endogenous mutant p53. When designing co-immunoprecipitation experiments with PPP1R13L antibodies, researchers must account for mixed tetramers of wild-type and mutant p53 .

• p53-independent p63 inhibition: In p53-mutant cells, PPP1R13L can still inhibit p63 independently of p53. This requires careful interpretation of antibody-based detection results to distinguish direct effects on p63 from those mediated through p53 .

• Modified validation protocols: For cells with p53 mutations, additional validation steps are needed when using PPP1R13L antibodies, including:

  • Parallel immunoprecipitation with both p53 and p63 antibodies

  • Controls with p53-null cells

  • Domain-specific antibodies to map interaction regions

These considerations are essential when studying cancers with high rates of p53 mutations, as PPP1R13L may exhibit context-dependent functions that vary based on the specific p53 mutation present .

What controls should be included when using PPP1R13L antibodies for research?

To ensure reliable results when using PPP1R13L antibodies, incorporate these essential controls:

• Positive tissue/cell controls:

  • Human heart tissue, HEK-293 cells, and mouse testis tissue have been validated as positive controls for Western blot

  • Human breast cancer tissue serves as a positive control for immunohistochemistry

• Knockdown/knockout validation:

  • Include PPP1R13L knockdown samples to confirm antibody specificity, as demonstrated in studies where PPP1R13L mRNA level was lower in patient-derived fibroblasts compared to controls

  • Western blot analysis confirming absence of the ~100 kDa band in PPP1R13L-deficient cells

• Peptide competition assays:

  • Pre-incubate antibody with the immunizing peptide (e.g., synthetic peptide corresponding to amino acids 780-797 of human iASPP isoform 1)

  • Signal should be specifically blocked by the competing peptide

• Isotype controls:

  • Include the appropriate isotype control (e.g., rabbit IgG for polyclonal antibodies or mouse IgG2b for monoclonal antibodies)

  • Use at the same concentration as the primary antibody

• Cross-validation with multiple antibodies:

  • Whenever possible, confirm results using antibodies targeting different epitopes of PPP1R13L to rule out non-specific binding

How can researchers address discrepancies in PPP1R13L detection between RNA and protein levels?

Discrepancies between PPP1R13L RNA and protein levels are common and can be addressed through several methodological approaches:

• Integrated RNA-protein correlation analysis:

  • RNA-protein correlations vary across genes and tissues, limiting the utility of RNA-based assays to predict protein expression

  • Perform parallel qRT-PCR and Western blot analyses to establish correlation patterns specific to your experimental system

• Post-translational modification considerations:

  • PPP1R13L is subject to post-translational modifications and proteolytic processing (e.g., Caspase-3 cleavage creating a stable 295-828aa fragment)

  • Use antibodies targeting different regions of the protein to detect full-length vs. processed forms

• Protein stability assessment:

  • PPP1R13L can affect p53 stability through enhanced proteasomal degradation

  • Incorporate proteasome inhibitors (e.g., MG132) in experiments to determine if protein stability, rather than transcription, explains the discrepancy

• Subcellular localization analysis:

  • The protein may relocalize between cellular compartments while total levels remain constant

  • Perform fractionation experiments or immunofluorescence with PPP1R13L antibodies to detect compartment-specific changes

• Paired RNA-protein assays:

  • When possible, derive information from paired RNA and proteomic assays from the same samples

  • Single-cell approaches combining RNA and protein detection may provide insights into cell-specific discrepancies

Research has shown that PPP1R13L can influence protein levels of targets like p53 through enhanced degradation rather than transcriptional changes , highlighting the importance of integrated RNA-protein analyses.

How can PPP1R13L antibodies be used to study its role in cardiac pathologies?

PPP1R13L antibodies are valuable tools for investigating the protein's role in cardiac disorders:

• Genetic cardiomyopathy studies: In Arab Christian infants diagnosed with dilated cardiomyopathy (DCM) associated with skin, teeth, and hair abnormalities, homozygous sequence variations creating premature stop codons in PPP1R13L were identified. Antibody-based methods confirmed the complete absence of iASPP protein in patient-derived fibroblasts .

• Inflammatory response analysis: PPP1R13L deficiency leads to hypersensitivity to lipopolysaccharide in an NF-κB-dependent manner. Antibodies can be used to study:

  • NF-κB binding activity to pro-inflammatory cytokine gene promoters

  • Correlation between PPP1R13L levels and cardiac inflammation

• Animal model validation: PPP1R13L antibodies can be employed in immunohistochemistry and Western blot analyses of heart tissues from Ppp1r13l-deficient mice to track DCM development stages .

• Therapeutic development: By establishing the crucial role of iASPP in dampening cardiac inflammatory responses, antibody-based screening could identify compounds that modulate PPP1R13L function as potential therapeutic targets for cardiac conditions .

What methodologies can be employed to study PPP1R13L's role in cancer progression and metastasis?

To investigate PPP1R13L's contribution to cancer progression, researchers can employ multiple antibody-based methodologies:

• In vivo tumor models: PPP1R13L overexpression strongly accelerates tumor formation by RAS/E1A transformed cells and produces a phenotype with multiple tumor nodes, consistent with increased metastasis. Use PPP1R13L antibodies to:

  • Track protein expression in primary vs. metastatic lesions

  • Monitor changes in PPP1R13L subcellular localization during metastatic progression

• Pathway analysis: PPP1R13L promotes cervical cancer progression through the PTEN/AKT/mTOR pathway. Antibody-based methods can assess:

  • Correlation between PPP1R13L levels and PTEN expression

  • Activation status of downstream AKT/mTOR signaling components

• Clinical correlation studies: In acute myeloid leukemia, high iASPP expression predicts poor outcomes independently of established risk classifications. Antibody-based tissue microarray analysis can help:

  • Stratify patients based on PPP1R13L expression levels

  • Identify correlations with treatment response and survival

• Mechanistic dissection: Using specific domain antibodies (e.g., against Ank-SH3 domain) can help elucidate how PPP1R13L interacts with p53, p63, and other partners to promote cancer progression .

• Therapeutic response prediction: PPP1R13L antibody staining intensity in pre-treatment biopsies might serve as a biomarker for predicting response to specific therapies, particularly those targeting apoptotic pathways or p53 family function .

What emerging technologies might enhance the utility of PPP1R13L antibodies in research?

Several cutting-edge technologies promise to expand the applications of PPP1R13L antibodies:

• Single-cell antibody screening approaches: New methods for simultaneously validating RNA-based predictions of multiple markers can be applied to PPP1R13L research. These approaches combine unique cell surface barcoding with experimental markers of interest and pooled analysis by methods like CyTOF .

• Proximity-dependent biotinylation (BioID/TurboID): Fusion of PPP1R13L with biotin ligases, followed by detection with specific antibodies, can map the proximal interactome of PPP1R13L in different cellular compartments and under various conditions.

• Antibody-based PROTAC development: Utilizing the specificity of PPP1R13L antibodies to design proteolysis-targeting chimeras (PROTACs) could enable selective degradation of PPP1R13L in cancer cells, potentially reversing oncogenic phenotypes identified in cervical cancer and AML studies .

• Multiplexed imaging technologies: Methods like CO-Detection by indEXing (CODEX) or Multiplexed Ion Beam Imaging (MIBI) would allow simultaneous detection of PPP1R13L alongside dozens of other proteins in tissue sections, providing spatial context to PPP1R13L interactions.

• Nanobody development: Generating single-domain antibodies (nanobodies) against PPP1R13L could enhance intracellular tracking and potentially interfere with specific protein-protein interactions involving distinct domains like the Ank-SH3 region .

What considerations should researchers keep in mind when designing antibody panels that include PPP1R13L?

When incorporating PPP1R13L antibodies into multiplex panels, researchers should consider:

• Epitope compatibility: Ensure that antibodies in the panel recognize distinct epitopes and won't sterically hinder each other's binding, particularly important when studying PPP1R13L interactions with p53 family members .

• Expression correlation validation: The utility of RNA-based assays to derive antibody panels is limited by RNA-protein correlations that vary across genes and tissues. Future approaches should incorporate explicit information on these correlations derived from paired RNA and proteomic assays .

• Signal separation considerations:

  • For flow cytometry or mass cytometry panels, select fluorophores or metal isotopes with minimal spillover

  • For immunofluorescence multiplexing, consider the subcellular localization pattern of PPP1R13L (nuclear vs. cytoplasmic) when pairing with other markers

• Functional marker integration: Include markers of pathways known to be regulated by PPP1R13L, such as:

  • p53 pathway members

  • NF-κB signaling components

  • PTEN/AKT/mTOR pathway elements

  • EMT markers

• Tissue context optimization: PPP1R13L is highly expressed in tissues such as the skin, esophagus, myocardium, vagina, and cervix. Panel design should account for tissue-specific expression patterns and potential background issues .