SESN3 Antibody

What is SESN3 and why is it important in research?

SESN3 (Sestrin 3) belongs to the sestrin protein family of stress-induced proteins with significant roles in cellular metabolism and stress responses. The human SESN3 protein has a canonical length of 492 amino acid residues with a molecular weight of approximately 57.3 kDa and is primarily localized in the cytoplasm . SESN3 functions as an intracellular leucine sensor that negatively regulates the TORC1 signaling pathway and activates AMP-activated protein kinase (AMPK), contributing to the suppression of mammalian target of rapamycin complex 1 (mTORC1) signaling . Its research importance stems from its roles in metabolic regulation, oxidative stress protection, and potential tumor suppression functions, particularly in liver diseases .

What species reactivity can be expected with commercial SESN3 antibodies?

Most commercially available SESN3 antibodies demonstrate reactivity with human samples, while many also cross-react with mouse and rat homologs due to high sequence conservation . When selecting an antibody for your research, verify the specific reactivity in product documentation, as some antibodies may have limited species reactivity. For example, several polyclonal antibodies like those cataloged under ab97792 from Abcam have been validated for reactivity with human, mouse, and rat samples . Always check validation data provided by manufacturers showing positive Western blot results in relevant tissue or cell line samples from your species of interest.

What are the common applications for SESN3 antibodies?

The most widely validated applications for SESN3 antibodies include:

The optimal dilution should be determined empirically for each experimental system and application .

How can I determine if my SESN3 antibody is detecting the correct isoforms?

SESN3 undergoes alternative splicing, yielding three different isoforms with varying molecular weights (approximately 36 kDa, 41 kDa, and 57 kDa) . To confirm isoform specificity:

• Run a Western blot analysis with proper molecular weight markers and look for bands at the expected sizes (particularly around 50-59 kDa for the canonical form)

• Include positive control lysates from tissues known to express SESN3 (e.g., liver tissue)

• Perform validation with recombinant SESN3 protein of known isoforms

• Consider using knockout/knockdown validation: compare samples from SESN3 knockout or knockdown models with wild-type controls to confirm antibody specificity

• For advanced validation, perform mass spectrometry analysis of immunoprecipitated proteins

Note that observed molecular weights may vary slightly from calculated weights due to post-translational modifications or experimental conditions .

What are the key considerations when investigating SESN3-mediated signaling pathways?

When studying SESN3-mediated signaling, researchers should consider:

• Pathway components: Focus on AMPK activation status, mTORC1/mTORC2 pathways, insulin receptor signaling, and TGFβ-Smad signaling pathway components

• Protein-protein interactions: Investigate interactions with:

  • mTOR and TSC2 (tuberous sclerosis complex 2)

  • Smad3 and Smad7 (for TGFβ pathway regulation)

  • Gli2 (for hedgehog signaling)

• Functional readouts: Monitor:

  • Phosphorylation status of direct targets (p-Akt, p-Stat3)

  • Expression of downstream genes (e.g., Pdgfrb, Cd44)

  • Metabolic parameters in relevant tissues

When designing experiments, use appropriate cellular stresses (oxidative stress, nutrient deprivation) to activate SESN3 function. Consider tissue-specific expression patterns, as SESN3 function may vary between tissues . For comprehensive pathway analysis, combine genetic approaches (SESN3 overexpression or knockdown) with pharmacological modifiers of relevant pathways.

How does SESN3 expression correlate with disease progression in hepatocellular carcinoma and other liver diseases?

Research indicates an important relationship between SESN3 expression and liver disease progression:

• Hepatocellular carcinoma (HCC):

  • TCGA dataset analysis reveals patients with higher hepatic SESN3 mRNA levels tend to have better survival rates

  • Sesn3 knockout mice develop more severe HCC with higher levels of alpha-fetoprotein, arginase 1, and cytokeratin 19

  • Sesn3 deficiency is associated with higher metastatic rates in experimental HCC models

• Nonalcoholic steatohepatitis (NASH):

  • Hepatic SESN3 protein is decreased by approximately 50% in NASH patients compared to controls

  • SESN3 staining intensity significantly decreases from non-fibrotic (F0) to fibrotic livers (F1-F4)

  • Sesn3 knockout mice develop severe NASH phenotype with only 4 weeks of dietary challenge

  • Sesn3 transgenic mice show protection against NASH development even after 8 weeks of NASH-inducing diet

• Mechanism of protection:

  • Sesn3 inhibits TGFβ receptor through interaction with Smad7

  • Direct inhibition of Smad3 function through protein-protein interaction and cytosolic retention

  • Suppression of hedgehog signaling through regulation of Gli2

These findings suggest SESN3 serves as a critical tumor suppressor and protective factor against liver diseases, making it a potential therapeutic target and prognostic marker .

What is the optimal protocol for detecting SESN3 by Western blotting?

For optimal Western blot detection of SESN3:

Sample Preparation:

• Extract proteins from cells or tissues using RIPA buffer containing protease inhibitors

• Determine protein concentration (Bradford or BCA assay)

• Load 20-30 μg of total protein per lane

Gel Electrophoresis and Transfer:

• Use 7.5-10% SDS-PAGE for better resolution of SESN3 (~57 kDa)

• Transfer to PVDF or nitrocellulose membrane (PVDF recommended for higher sensitivity)

Antibody Incubation:

• Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Incubate with primary SESN3 antibody at dilutions between 1:500-1:1000 overnight at 4°C

• Wash 3x with TBST (10 minutes each)

• Incubate with HRP-conjugated secondary antibody (typically 1:5000-1:10000) for 1 hour at room temperature

• Wash 3x with TBST (10 minutes each)

Detection:

• Use enhanced chemiluminescence (ECL) detection system

• Expected bands around 50-59 kDa for full-length SESN3

• Multiple bands may be observed due to alternative splicing isoforms (~36 kDa, ~51-59 kDa)

Controls:

• Include positive controls like HeLa, A431, HEK-293, HepG2 cells, or mouse liver tissue

• Consider using SESN3 knockout samples as negative controls

What are the recommended protocols for immunofluorescence staining of SESN3?

For immunofluorescence detection of SESN3:

Cell Preparation:

• Culture cells on coverslips or chamber slides to 60-80% confluence

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

Tissue Section Preparation:

• For paraffin sections, deparaffinize and rehydrate

• Perform antigen retrieval using 1mM EDTA buffer (pH 8.0) at 100°C for 5 minutes

• Allow sections to cool to room temperature

Antibody Staining:

• Block with 5% normal serum (e.g., horse serum) for 1 hour at room temperature

• Incubate with SESN3 primary antibody (1:200 dilution) overnight at 4°C

• Wash 3x with PBS (5 minutes each)

• Incubate with fluorophore-conjugated secondary antibody (1:500) for 1 hour at room temperature in the dark

• Wash 3x with PBS (5 minutes each)

• Counterstain nuclei with DAPI (1:1000) for 5 minutes

• Mount with anti-fade mounting medium

Imaging:

• Observe under confocal microscope for optimal resolution

• Expected cytoplasmic localization of SESN3

• For co-localization studies, consider double staining with markers of interest

Validated cell lines include A549 (human lung carcinoma), which show clear cytoplasmic localization of SESN3 .

What methods should be used to study SESN3-protein interactions in research settings?

To study SESN3-protein interactions, researchers can employ these methodologies:

Co-Immunoprecipitation (Co-IP):

• Lyse cells in a non-denaturing buffer to preserve protein-protein interactions

• Pre-clear lysate with protein A/G beads

• Incubate with SESN3 antibody or antibody against suspected interacting protein

• Precipitate using protein A/G beads

• Analyze by Western blot for co-precipitated proteins

This approach has been successfully used to characterize SESN3 interactions with Smad7, Smad3, and other signaling proteins .

Recombinant Protein Expression and Pull-down Assays:

• Generate constructs with tagged SESN3 (e.g., FLAG or HA tag)

• Express in suitable system (e.g., HEK 293 cells)

• Perform pull-down using anti-tag antibodies

• Analyze interacting proteins by Western blot or mass spectrometry

Fluorescence Resonance Energy Transfer (FRET):

• Generate fluorescent protein fusions (e.g., SESN3-GFP and interacting protein-RFP)

• Express in cells and monitor FRET signal

• Use acceptor photobleaching or sensitized emission to confirm interactions

Proximity Ligation Assay (PLA):

• Fix and permeabilize cells/tissues

• Incubate with primary antibodies against SESN3 and suspected interacting protein

• Apply PLA probes and perform ligation and amplification

• Visualize interaction-specific signals by fluorescence microscopy

Bimolecular Fluorescence Complementation (BiFC):

• Generate split fluorescent protein constructs fused to SESN3 and potential interacting proteins

• Co-express in cells and monitor reconstituted fluorescence

These methods have been successfully applied to identify and characterize interactions between SESN3 and components of the TGFβ-Smad pathway, hedgehog signaling, and other metabolic regulatory proteins .

How can I resolve common issues with SESN3 antibody specificity in Western blotting?

Researchers encountering specificity issues with SESN3 antibodies should consider these troubleshooting approaches:

For definitive validation, consider using SESN3 knockout or knockdown samples as negative controls and recombinant SESN3 protein as a positive control .

What are the best practices for validating a new SESN3 antibody for research applications?

When validating a new SESN3 antibody, follow these comprehensive steps:

• Western Blot Validation:

  • Test antibody at multiple dilutions (1:200, 1:500, 1:1000, 1:2000)

  • Use positive control lysates from various tissues/cells known to express SESN3 (e.g., liver, HeLa, HEK293)

  • Include negative controls (SESN3 knockout/knockdown samples if available)

  • Verify expected molecular weight bands (57 kDa canonical form, plus possible 36-41 kDa isoforms)

• Immunohistochemistry/Immunofluorescence Validation:

  • Test on both formalin-fixed paraffin-embedded and frozen tissues

  • Compare staining pattern to published literature (cytoplasmic localization)

  • Perform antigen retrieval optimization

  • Include appropriate isotype controls

• Advanced Validation Approaches:

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide before application

  • Genetic validation: Compare staining in wild-type vs. SESN3 knockout/knockdown samples

  • Orthogonal validation: Compare protein detection with mRNA expression data

  • Independent antibody validation: Compare results with a second antibody targeting a different epitope

• Application-Specific Validation:

  • For each application (WB, IHC, IF, IP, ELISA), perform separate validation tests

  • Document optimal conditions for each application

  • Determine the detection limits for quantitative applications

Following these validation steps ensures reliability of research findings and enhances reproducibility across different experimental conditions.

How should researchers interpret contradictory findings when using different SESN3 antibodies?

When confronted with contradictory results using different SESN3 antibodies:

• Compare antibody characteristics:

  • Epitope locations: Antibodies targeting different domains may detect different isoforms

  • Clonality: Monoclonal vs. polyclonal antibodies have different specificity profiles

  • Host species: Different hosts may produce antibodies with varying affinities

  • Validation history: Check if antibodies have been cited in peer-reviewed publications

• Evaluate experimental conditions:

  • Tissue/cell-specific expression patterns may vary

  • Different fixation/extraction methods can affect epitope accessibility

  • Post-translational modifications may mask certain epitopes

• Verification approaches:

  • Perform siRNA/shRNA knockdown or CRISPR knockout experiments to verify specificity

  • Use recombinant expression of SESN3 with tags for independent detection

  • Employ orthogonal detection methods (e.g., mass spectrometry)

  • Check for batch-to-batch variations using lot numbers

• Data integration strategy:

  • Prioritize results from antibodies with published validation data

  • Consider the consensus findings across multiple antibodies

  • Report discrepancies transparently in publications

  • When possible, use complementary genetic approaches to confirm findings

What emerging technologies are enhancing SESN3 research beyond traditional antibody applications?

Several cutting-edge technologies are advancing SESN3 research beyond conventional antibody-based methods:

• CRISPR/Cas9 Gene Editing:

  • Generation of SESN3 knockout models for mechanistic studies

  • Creation of endogenously tagged SESN3 (e.g., GFP-SESN3) for live-cell imaging

  • Precise introduction of disease-relevant mutations to study structure-function relationships

• Proximity-Dependent Labeling:

  • BioID or APEX2 fusion to SESN3 to identify the complete interactome

  • TurboID for rapid labeling of transient interactions in specific cellular compartments

  • Helps identify novel binding partners beyond known interactions with mTOR, Smad3, and Smad7

• Advanced Imaging Technologies:

  • Super-resolution microscopy for detailed subcellular localization

  • Live-cell imaging with optogenetic control of SESN3 activity

  • FRET-based biosensors to monitor SESN3-protein interactions in real-time

• Single-Cell Technologies:

  • Single-cell RNA-seq to map SESN3 expression patterns across diverse cell populations

  • Single-cell proteomics to quantify SESN3 protein levels in heterogeneous samples

  • Spatial transcriptomics to visualize SESN3 expression within tissue architecture

• Structural Biology Approaches:

  • Cryo-EM studies of SESN3 complexes with interacting partners

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • In silico molecular dynamics simulations to predict functional domains

These technologies are collectively enabling researchers to develop a more comprehensive understanding of SESN3 function in health and disease states.

How can researchers effectively study the differential roles of SESN3 isoforms?

To investigate the distinct functions of SESN3 isoforms, researchers should implement these specialized approaches:

• Isoform-Specific Detection:

  • Design PCR primers spanning exon junctions unique to specific isoforms

  • Generate isoform-specific antibodies targeting unique peptide sequences

  • Use RNA-seq with isoform quantification algorithms to measure relative abundance

• Isoform-Selective Manipulation:

  • Design siRNAs or shRNAs targeting unique exons or exon junctions

  • Use CRISPR-Cas9 with guide RNAs directed at isoform-specific exons

  • Create isoform-selective knockout cell lines or animal models

• Expression Systems:

  • Clone individual SESN3 isoforms into expression vectors

  • Generate stable cell lines expressing single isoforms

  • Use inducible expression systems to control timing and levels of expression

• Functional Characterization:

  • Compare subcellular localization patterns of different isoforms

  • Assess ability of individual isoforms to interact with known SESN3 partners

  • Evaluate differential effects on signaling pathways (mTOR, AMPK, TGFβ)

  • Measure metabolic parameters affected by each isoform

• Disease-Relevance Studies:

  • Analyze isoform expression ratios in disease samples vs. normal tissues

  • Correlate isoform levels with disease progression or outcome measures

  • Determine if specific disease states alter splicing patterns of SESN3

These methods will help unravel the potentially distinct functions of the three reported SESN3 isoforms (36 kDa, 41 kDa, and 57 kDa) and their specific roles in cellular homeostasis, stress response, and disease pathogenesis.