Recombinant Bovine E3 ubiquitin-protein ligase TRIM13 (TRIM13)

What is TRIM13 and what are its key structural characteristics?

TRIM13 (Tripartite Motif Containing Protein 13) is an E3 ubiquitin-protein ligase that belongs to the TRIM family of proteins. Bovine TRIM13 consists of 407 amino acids and contains several conserved domains: an N-terminal RING (R) domain, a B-Box (B) domain, a coiled-coil (CC) domain, and a C-terminal transmembrane (TM) domain. The RING domain is essential for its E3 ligase activity, while the TM domain anchors it to membranes, particularly the endoplasmic reticulum. The protein's amino acid sequence includes multiple functional motifs that enable protein-protein interactions and substrate recognition for ubiquitination .

What are the known biological functions of TRIM13?

TRIM13 serves multiple biological functions:

• Cell cycle regulation: Nuclear-localized TRIM13 drives cell cycle entry through stabilization of cyclin A1, thereby repressing self-renewal in certain cell types, particularly in leukemic cells .

• Immune regulation: TRIM13 modulates DNA-triggered inflammatory cytokine production and inhibits DNA virus replication through interaction with the STING protein at the endoplasmic reticulum membrane .

• Tumor suppression: TRIM13 (also known as RFP2) functions as a putative tumor suppressor, with its expression often downregulated in certain cancers .

• Inflammatory regulation: TRIM13 deficiency has been linked to age-related autoinflammation, suggesting a role in maintaining immune homeostasis and preventing aberrant inflammation .

• Transcriptional regulation: TRIM13 expression is directly regulated by chromatin factors such as CHAF1B, indicating its involvement in epigenetic control mechanisms .

How should recombinant TRIM13 protein be reconstituted and stored for optimal activity?

For optimal activity of recombinant bovine TRIM13 protein, follow these methodological guidelines:

• Initial preparation: Briefly centrifuge the vial prior to opening to bring the contents to the bottom.

• Reconstitution: Reconstitute the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• Glycerol addition: Add glycerol to a final concentration of 5-50% (recommended default: 50%) to maintain protein stability during freeze-thaw cycles.

• Storage conditions: Store at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use to avoid repeated freeze-thaw cycles.

• Short-term storage: Working aliquots can be stored at 4°C for up to one week.

• Buffer conditions: The protein is supplied in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0, which helps maintain stability .

Avoiding repeated freeze-thaw cycles is critical as they can significantly reduce protein activity. For experiments requiring multiple uses over time, creating single-use aliquots immediately after reconstitution is strongly recommended to preserve functionality.

What are the recommended methods for CRISPR-mediated TRIM13 knockout in experimental systems?

Based on successful experimental approaches documented in research, CRISPR-mediated TRIM13 knockout can be achieved through the following methodology:

• Guide RNA design: Design single guide RNAs targeting early in the coding sequence of TRIM13, particularly focusing on the RING domain for functional studies without complete protein elimination.

• Cas9 complex formation: Complex the guide RNAs with HiFi Cas9 V3 (or equivalent) for approximately 20 minutes at room temperature before resuspending in appropriate electroporation buffer with Cas9 Electroporation Enhancer.

• Cell electroporation parameters:

  • For human AML cells: 1350 V, 35 ms, 1 pulse

  • For mouse AML cells: 1700 V, 20 ms, 1 pulse

• Post-electroporation handling: Plate cells in complete media without antibiotics for 24 hours and assess CRISPR editing efficiency after 48 hours via Sanger sequencing and inference of CRISPR edits analysis.

• Clone selection: Plate cells in methylcellulose and select single colonies for expansion. Confirm TRIM13 knockout in candidate colonies first via Sanger sequencing, followed by western blot validation.

• Characterization: Distinguish between in-frame deletions (RINGdel) or out-of-frame knockouts (complete TRIM13 knockout) via sequencing and protein expression analysis .

This approach allows for generation of either specific domain deletions or complete knockout models, enabling functional characterization of different TRIM13 domains.

How can researchers effectively detect and quantify TRIM13 expression at protein and mRNA levels?

Effective detection and quantification of TRIM13 can be achieved through multiple complementary approaches:

Protein Level Detection:

• Western blotting: Use specific antibodies against TRIM13 or against the His-tag if working with recombinant His-tagged TRIM13. For bovine TRIM13, verify antibody cross-reactivity before experimentation.

• Immunofluorescence: For subcellular localization studies, fixed cells can be stained with TRIM13-specific antibodies and visualized using confocal microscopy. Co-staining with ER markers (for transmembrane localization) or nuclear markers provides additional context for localization studies.

• Co-immunoprecipitation (Co-IP): For interaction studies, TRIM13 can be immunoprecipitated using specific antibodies and interacting partners can be detected by western blotting.

mRNA Level Detection:

• Isoform-specific quantification: Use Taqman quantitative PCR with probes that exclusively recognize specific isoforms (e.g., 201 or 202 isoforms of TRIM13) to differentiate expression from different promoters.

• RNA sequencing: For comprehensive transcriptome analysis, RNA-seq can identify TRIM13 expression levels and alternative promoter usage.

• Chromatin immunoprecipitation sequencing (ChIP-seq): To study transcriptional regulation of TRIM13, ChIP-seq with antibodies against transcription factors or histone modifications (e.g., H3K4me3) can identify regulatory regions .

These methodologies can be combined to provide a comprehensive understanding of TRIM13 expression patterns and regulatory mechanisms in different experimental contexts.

How does TRIM13 interact with the STING pathway, and what experimental approaches can elucidate this interaction?

TRIM13 interacts with STING (Stimulator of Interferon Genes) primarily through their transmembrane domains, forming a functional complex at the endoplasmic reticulum membrane. This interaction plays a critical role in regulating DNA-triggered immune responses.

Experimental approaches to elucidate this interaction include:

• Domain mapping through truncation mutants:

  • Create constructs expressing full-length TRIM13, TRIM13 without RING domain, TRIM13 without TM domain, and TRIM13 with only RING domain

  • Similarly, generate full-length STING, STING with only TM domain, and STING without TM domain

  • Perform co-immunoprecipitation assays with these mutants to identify interacting domains

• Colocalization studies using confocal microscopy:

  • Express fluorescently tagged TRIM13 and STING in cells

  • Examine colocalization patterns under resting conditions and after stimulation (e.g., HSV-1 infection)

  • Quantify colocalization coefficients to measure interaction strength

• Functional assays to assess regulatory effects:

  • Measure interferon production in TRIM13-deficient cells with reconstituted wild-type or domain-mutant TRIM13

  • Assess STING-dependent signaling pathway activation through phosphorylation of downstream effectors like TBK1 and IRF3

Research has shown that DNA virus infection can attenuate the colocalization of TRIM13 with STING, suggesting dynamic regulation of this interaction during immune responses. The TM domains of both proteins are essential for their interaction, as deletion of the TM domain in either protein abolishes the interaction .

What is the relationship between TRIM13 and CHAF1B in transcriptional regulation, and how can this be studied?

The relationship between TRIM13 and CHAF1B (Chromatin Assembly Factor 1 Subunit B) represents a critical regulatory mechanism in transcriptional control, particularly in leukemic cells:

Regulatory Relationship:
CHAF1B functions as a transcriptional repressor of TRIM13 by binding to its promoter region. In leukemic cells, CHAF1B overexpression leads to TRIM13 repression, which enhances self-renewal capacity. Conversely, CHAF1B loss results in increased TRIM13 expression through alternative promoter usage, driving cell cycle entry and reduced self-renewal.

Experimental Approaches to Study This Relationship:

• Chromatin Occupancy Analysis:

  • Chromatin immunoprecipitation (ChIP) assays to detect CHAF1B binding at the TRIM13 promoter

  • POLR2A ChIP to identify active transcription sites at different TRIM13 promoters

  • H3K4me3 ChIP to identify active promoter regions

  • Assay for transposase-accessible chromatin (ATAC-seq) to assess chromatin accessibility at TRIM13 promoters

• Isoform-Specific Expression Analysis:

  • Design Taqman probes specific to different TRIM13 isoforms (e.g., Trim13-201 vs. Trim13-202)

  • Quantify isoform-specific expression under conditions of CHAF1B overexpression or knockdown

• Functional Reporter Assays:

  • Clone the TRIM13 promoter sequence bound by CHAF1B into a promoterless vector driving luciferase expression

  • Co-transfect with CHAF1B expression constructs (wild-type or binding-deficient mutants like CHAF1B RRAA)

  • Measure luciferase activity to quantify transcriptional repression

• Manipulation of Alternative Promoters:

  • Use CRISPR-based approaches to delete specific TRIM13 promoters (proximal or distal)

  • Assess the impact on TRIM13 expression and cellular phenotypes under conditions of CHAF1B presence or absence

• Cellular Consequence Assessment:

  • Colony formation assays with CD34+ peripheral blood stem cells with combined CHAF1B overexpression and TRIM13 knockdown

  • Cell cycle analysis in AML cell lines (e.g., U937, MOLM13) after CHAF1B manipulation

These approaches collectively provide insights into the molecular mechanisms by which CHAF1B regulates TRIM13 expression and the functional consequences of this regulation in normal and malignant hematopoiesis.

What is the role of TRIM13 in age-related autoinflammation, and how can this be modeled experimentally?

TRIM13 plays a significant role in preventing age-related autoinflammation, with its deficiency leading to progressive inflammatory manifestations in multiple organs. This function appears linked to its regulation of DNA-triggered immune responses.

Experimental Approaches to Model and Study TRIM13's Role in Autoinflammation:

• Generation of Age-Dependent Mouse Models:

  • Create TRIM13-deficient mice (Trim13^-/-) through genetic approaches

  • Maintain cohorts to different age points (e.g., 5 months vs. 10 months)

  • Compare inflammatory phenotypes between age groups to establish age-dependency

• Histological Examination of Multiple Organs:

  • Perform H&E staining of tissues from lungs, liver, kidney, heart, and brain

  • Quantify inflammatory cell infiltration in each organ system

  • Compare patterns across different ages and between wild-type and TRIM13-deficient animals

• Cytokine Profiling:

  • Measure serum levels of inflammatory cytokines such as IFNβ and IL-6 using ELISA

  • Perform qPCR assays to quantify Ifnb and Il6 expression in affected tissues

  • Track changes in cytokine levels with age progression

• Mechanistic Investigation:

  • Examine the impact of TRIM13 deficiency on STING pathway activation

  • Analyze potential accumulation of cytosolic DNA with age

  • Investigate whether crossing TRIM13-deficient mice with STING-deficient mice rescues the inflammatory phenotype

• Translational Relevance:

  • Analyze TRIM13 expression in human samples from patients with autoinflammatory conditions

  • Correlate TRIM13 expression levels with disease severity and age of onset

Research has shown that while 5-month-old Trim13^-/- mice show minimal inflammatory signs, 10-month-old Trim13^-/- mice develop extensive inflammatory cell infiltrations in multiple organs, with the highest prevalence in the lung (87.5% of mice), kidney (75.0%), and liver (50.0%). This is accompanied by significantly increased levels of IFNβ and IL-6 in the serum, suggesting systemic inflammation .

This age-dependent progression makes TRIM13-deficient mice an excellent model for studying mechanisms of age-related autoinflammation and testing potential therapeutic interventions.

What are common challenges in working with recombinant TRIM13 protein, and how can they be addressed?

Researchers working with recombinant TRIM13 protein often encounter several challenges. Here are the most common issues and their solutions:

When working with recombinant TRIM13, it's essential to validate its functional activity before experimental use, particularly when investigating its E3 ligase activity or protein-protein interactions.

How can researchers differentiate between the functions of different TRIM13 domains in experimental settings?

Differentiating between the functions of TRIM13's domains requires strategic experimental approaches that isolate domain-specific activities while controlling for other variables. Here's a methodological framework:

Research has demonstrated that the TM domain of TRIM13 is essential for interaction with STING, while the RING domain is likely responsible for its E3 ligase activity towards specific substrates like cyclin A1. Understanding domain-specific functions can provide insights into how TRIM13 simultaneously regulates multiple cellular processes .

What approaches can be used to study TRIM13 isoform-specific expression and function?

TRIM13 is expressed through multiple isoforms that may have distinct functions. Studying isoform-specific expression and function requires specialized approaches:

• Isoform Identification and Characterization:

  • RNA-seq analysis to identify all expressed TRIM13 isoforms

  • 5' RACE (Rapid Amplification of cDNA Ends) to precisely map transcription start sites

  • Cloning and sequencing of full-length cDNAs for each isoform

• Quantitative Isoform-Specific Expression Analysis:

  • Design of isoform-specific primers/probes that span unique exon junctions

  • Taqman quantitative PCR with probes that exclusively recognize specific isoforms (e.g., Trim13-201 vs. Trim13-202)

  • Digital droplet PCR for absolute quantification of low-abundance isoforms

• Promoter-Specific Regulation Studies:

  • Chromatin immunoprecipitation (ChIP) to identify transcription factor binding at alternative promoters

  • POLR2A ChIP to determine RNA polymerase II occupancy at different promoters

  • H3K4me3 ChIP and ATAC-seq to assess chromatin accessibility at alternative promoters

• Isoform-Specific Functional Analysis:

  • CRISPR-mediated deletion of specific exons or promoter regions

  • Selective knockdown of specific isoforms using siRNAs targeting unique regions

  • Ectopic expression of individual isoforms in knockout backgrounds

• Isoform-Specific Interaction Studies:

  • Co-immunoprecipitation assays with isoform-specific antibodies

  • Proximity labeling approaches (BioID, APEX) with isoform-specific constructs

  • Yeast two-hybrid screening with different isoforms as bait

Research has shown that in certain contexts, TRIM13 can be transcribed from either proximal (Trim13-201) or distal (Trim13-202) exons, with differential regulation by factors like CHAF1B. The choice of promoter can significantly impact protein expression levels and potentially function. In Chaf1b+/+ leukemic cells, TRIM13 is transcribed exclusively from the proximal exon, while Chaf1b deletion allows transcription from either the proximal or distal exon .

This understanding of isoform-specific expression is crucial for accurately interpreting TRIM13's role in different cellular contexts and disease states.

How is TRIM13 implicated in cancer biology, and what experimental models are most appropriate for studying its tumor suppressor function?

TRIM13 (also known as RFP2) functions as a putative tumor suppressor, with significant implications for cancer biology:

Role in Cancer:

• Leukemia: TRIM13 represses self-renewal in leukemic cells by driving cell cycle entry through stabilization of cyclin A1. Low TRIM13 expression correlates with enhanced leukemic cell self-renewal and is associated with poor prognosis.

• Transcriptional regulation: CHAF1B directly represses TRIM13 transcription, creating a regulatory axis that protects leukemic cell self-renewal. High CHAF1B and low TRIM13 expression pattern has been observed in AML samples, suggesting this relationship may be clinically relevant.

• Cell cycle control: Nuclear-localized TRIM13 influences cell proliferation through regulation of cell cycle proteins, potentially restricting uncontrolled growth in normal cells.

Appropriate Experimental Models:

• Cell line models:

  • AML cell lines (e.g., U937, MOLM13) for studying leukemia-specific functions

  • Paired isogenic cell lines with TRIM13 knockout, knockdown, or overexpression

  • Reporter cell lines to monitor cell cycle progression and self-renewal capacity

• Primary cell models:

  • CD34+ peripheral blood stem cells from healthy donors with manipulated TRIM13 expression

  • Patient-derived AML samples with varying levels of endogenous TRIM13 expression

  • Colony formation assays to assess self-renewal and differentiation capacity

• In vivo models:

  • TRIM13 knockout mice crossed with cancer-prone genetic backgrounds

  • Xenograft models using TRIM13-manipulated human cancer cells

  • Patient-derived xenografts with characterized TRIM13 expression levels

• Functional assays:

  • Colony formation assays to assess self-renewal capacity

  • Cell cycle analysis using flow cytometry

  • Long-term culture-initiating cell assays for stem cell function

Research has demonstrated that simultaneous CHAF1B overexpression and TRIM13 repression significantly enhanced colony formation capacity in CD34+ peripheral blood stem cells from healthy donors, suggesting this pathway plays a fundamental role in regulating normal and malignant hematopoiesis .

What is the relationship between TRIM13 and viral infections, and how can researchers study its antiviral mechanisms?

TRIM13 plays a significant role in antiviral immunity, particularly against DNA viruses, through its interaction with the STING pathway:

Antiviral Functions:

• TRIM13 deficiency enhances pathogenic-DNA-triggered inflammatory cytokine production

• TRIM13 inhibits DNA virus replication through regulation of innate immune responses

• TRIM13 interacts with STING at the endoplasmic reticulum membrane to modulate antiviral signaling

Experimental Approaches to Study Antiviral Mechanisms:

• Virus Infection Models:

  • DNA virus systems (e.g., HSV-1, vaccinia virus) in TRIM13-deficient or overexpressing cells

  • Measurement of viral replication kinetics using plaque assays or qPCR for viral genomes

  • Assessment of virus-induced cytopathic effects in the presence or absence of TRIM13

• STING Pathway Analysis:

  • Examination of STING-dependent signaling events (TBK1/IRF3 phosphorylation, type I interferon production)

  • Analysis of cGAS-STING pathway activation using reporter assays

  • Comparison of wild-type vs. TRIM13-deficient cells in response to synthetic STING agonists

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation of TRIM13 and STING before and after viral infection

  • Analysis of how viral infection affects TRIM13-STING colocalization using confocal microscopy

  • Domain mapping to identify regions critical for antiviral function

• Ubiquitination Analyses:

  • In vitro and in vivo ubiquitination assays to identify TRIM13 substrates during viral infection

  • Mass spectrometry to identify ubiquitination sites on potential targets

  • Functional consequences of ubiquitination on target protein stability or activity

• Transcriptional Profiling:

  • RNA-seq analysis of TRIM13-deficient vs. wild-type cells during viral infection

  • Identification of TRIM13-dependent gene expression programs

  • Integration with ChIP-seq data to identify direct vs. indirect effects

Research has shown that HSV-1 infection attenuates the colocalization of TRIM13 with STING, suggesting dynamic regulation of this interaction during viral infection. This interaction primarily occurs through the transmembrane domains of both proteins and appears to be a critical regulatory node in antiviral immunity .

How does TRIM13 contribute to inflammatory regulation, and what are the implications for autoimmune and inflammatory diseases?

TRIM13 plays a crucial role in inflammatory regulation, with its dysfunction linked to autoimmune and inflammatory conditions:

Inflammatory Regulatory Functions:

• Prevention of age-related autoinflammation: TRIM13-deficient mice develop progressive inflammatory cell infiltrations in multiple organs as they age, particularly in the lung, liver, and kidney.

• Regulation of cytokine production: TRIM13 deficiency leads to enhanced production of inflammatory cytokines like IFNβ and IL-6.

• Control of DNA-sensing pathways: Through interaction with STING, TRIM13 modulates responses to cytosolic DNA, a potent trigger of inflammation.

Experimental Approaches and Disease Implications:

Research has shown that 10-month-old TRIM13-deficient mice exhibit extensive inflammatory cell infiltration in multiple organs, with 87.5% showing infiltration in the lung, 75.0% in the kidney, and 50.0% in the liver. This was accompanied by significantly elevated levels of IFNβ and IL-6 in the serum, indicating systemic inflammation .

The connection between TRIM13, STING signaling, and inflammation suggests that TRIM13 dysfunction may contribute to human autoimmune and inflammatory diseases, particularly those characterized by aberrant type I interferon responses or age-related progression. Understanding these mechanisms could lead to novel therapeutic approaches for conditions like lupus, inflammatory myopathies, or interferonopathies.

What are the most promising directions for future TRIM13 research based on current findings?

Based on current knowledge about TRIM13's multi-faceted functions, several promising research directions emerge:

• Therapeutic Targeting of TRIM13 in Cancer:

  • Development of small molecules to modulate TRIM13 activity

  • Investigation of TRIM13 as a biomarker for leukemia and other cancers

  • Exploration of the CHAF1B-TRIM13 axis as a targetable pathway in malignancies

• TRIM13 in Age-Related Inflammatory Conditions:

  • Detailed characterization of age-dependent inflammatory phenotypes in TRIM13-deficient models

  • Identification of genetic or environmental factors that influence TRIM13-associated inflammation

  • Development of TRIM13-based therapeutic strategies for age-related inflammatory diseases

• Structural Biology of TRIM13:

  • Determination of high-resolution structures of TRIM13 domains

  • Analysis of TRIM13-substrate complexes

  • Structure-based drug design targeting specific TRIM13 functions

• TRIM13 in the STING Signaling Network:

  • Comprehensive mapping of TRIM13's role in the cGAS-STING pathway

  • Analysis of TRIM13's influence on STING trafficking and activation

  • Exploration of TRIM13 as a regulator of other innate immune pathways

• Post-Translational Modifications of TRIM13:

  • Identification of sites and types of modifications on TRIM13

  • Understanding how these modifications regulate TRIM13 function

  • Cross-talk between TRIM13's own ubiquitination and its E3 ligase activity

• TRIM13 Isoform-Specific Functions:

  • Comprehensive characterization of all TRIM13 isoforms across tissues

  • Analysis of isoform-specific interactomes and activities

  • Investigation of differential regulation of TRIM13 isoforms in disease states

These research directions build upon established findings regarding TRIM13's roles in cancer biology, inflammatory regulation, and antiviral immunity, while exploring new frontiers in structural biology, post-translational regulation, and therapeutic applications .

How do post-translational modifications affect TRIM13 function and how can they be experimentally analyzed?

As an E3 ubiquitin ligase, TRIM13 not only modifies other proteins but is also subject to various post-translational modifications (PTMs) that regulate its activity, localization, and stability. Understanding these modifications is crucial for comprehending TRIM13's complex functions.

Types of Post-Translational Modifications Affecting TRIM13:

• Ubiquitination: As an E3 ligase, TRIM13 can undergo auto-ubiquitination or be targeted by other E3 ligases

• Phosphorylation: Likely occurs on serine, threonine, or tyrosine residues in response to various signaling events

• SUMOylation: May regulate TRIM13's nuclear functions or protein-protein interactions

• Acetylation: Could affect TRIM13's binding to DNA or other proteins, particularly in nuclear contexts

• Methylation: May influence TRIM13's protein-protein interactions or subcellular localization

Experimental Approaches for PTM Analysis:

Understanding TRIM13's post-translational modifications will provide insights into how this protein's multiple functions are regulated in different cellular contexts and how these regulations may be altered in disease states.

What techniques are emerging for studying the dynamic interactions of TRIM13 with its binding partners in real-time cellular contexts?

Studying the dynamic interactions of TRIM13 with its binding partners in real-time cellular contexts requires advanced techniques that go beyond traditional biochemical approaches. These emerging methodologies provide unprecedented insights into protein interactions within their native environment:

• Live Cell Imaging Techniques:

  • Fluorescence Resonance Energy Transfer (FRET): Tag TRIM13 and potential partners with appropriate fluorophore pairs to detect interactions within 10 nm range

  • Bioluminescence Resonance Energy Transfer (BRET): Similar to FRET but using luciferase and fluorescent protein pairs, reducing photobleaching concerns

  • Split Fluorescent/Luciferase Complementation: Fuse complementary fragments to TRIM13 and interaction partners, which reconstitute activity upon interaction

• Proximity-Based Labeling Methods:

  • BioID: Fuse TRIM13 to a promiscuous biotin ligase that biotinylates proteins in close proximity

  • APEX2: Couple TRIM13 to engineered ascorbate peroxidase to catalyze biotin-phenol labeling of nearby proteins

  • TurboID: An evolved, faster version of BioID for temporal studies of TRIM13 interactomes

  • Split-BioID: Use complementary fragments of biotin ligase fused to potential interaction partners

• Super-Resolution Microscopy:

  • Stimulated Emission Depletion (STED) Microscopy: Visualize TRIM13 complexes below the diffraction limit

  • Photoactivated Localization Microscopy (PALM): Track single-molecule interactions of TRIM13

  • Stochastic Optical Reconstruction Microscopy (STORM): Map TRIM13 distribution and interactions with nanometer precision

• Real-Time Interaction Monitoring:

  • Lattice Light-Sheet Microscopy: Monitor TRIM13 interactions with reduced phototoxicity over extended periods

  • Single-Particle Tracking: Follow individual TRIM13 molecules to assess binding kinetics and residence times

  • Fluorescence Correlation Spectroscopy (FCS): Measure diffusion rates of TRIM13 complexes in living cells

• Optogenetic Approaches:

  • Light-inducible dimerization systems: Control TRIM13 interactions with temporal and spatial precision

  • Optogenetic activation of TRIM13 targets: Assess downstream effects of specific interactions