SESN2 Antibody, HRP conjugated

What is SESN2 and why is it an important research target?

SESN2 (Sestrin2) is a stress-inducible metabolic regulator that responds to diverse cellular stresses. It plays critical roles in:

• Inhibiting mTORC1 signaling through the GATOR complex as an intracellular leucine sensor

• Protecting against oxidative and genotoxic stresses

• Mediating mitophagy and mitochondrial health

• Regulating cellular metabolism through AMPK pathways

• Dampening aerobic glycolysis in glucose-limiting conditions

SESN2 is widely expressed across multiple tissues and primarily localized in the cytoplasm, with a canonical protein length of 480 amino acid residues and a mass of approximately 54.5 kDa . Its involvement in multiple stress response pathways makes it a valuable target for researchers studying cellular stress, metabolism, and disease states.

What are the expected molecular weights when detecting SESN2 with antibodies?

When using SESN2 antibodies in Western blot applications, researchers should expect:

• The canonical molecular weight of 54-60 kDa for the full-length protein

• Potential variations in observed weights (54-60 kDa range) depending on post-translational modifications

• In some cellular stress conditions, alternative bands may be observed

This information is critical for proper identification of SESN2 in experimental samples. Always include positive controls such as HEK-293, HeLa, or K-562 cell lysates, which have been confirmed to express detectable levels of SESN2 .

What are the optimal conditions for using HRP-conjugated SESN2 antibodies in Western blotting?

For optimal Western blot results with HRP-conjugated SESN2 antibodies:

When studying SESN2 interactions with binding partners like IGF2BP3 or HK2 mRNA, extended exposure times may be necessary to detect complex formation . For stress-induced upregulation experiments, ensure adequate sample collection time points as SESN2 expression increases substantially after 12 hours of LPS stimulation .

How should researchers validate SESN2 antibody specificity in their experimental systems?

Rigorous validation of SESN2 antibodies is essential to ensure experimental reliability:

• Genetic controls: Use SESN2 knockout (Sesn2^-/-) samples as negative controls

• Overexpression controls: Include SESN2-overexpressing samples (e.g., cells transfected with plasmid carrying the SESN2 gene)

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to confirm specific binding

• Cross-reactivity testing: Test across multiple species if working with non-human models

• Multiple antibody validation: Compare results from antibodies targeting different epitopes of SESN2

Validation data should be documented and included in publications. For example, research has shown that SESN2 antibodies can reliably detect the induced expression in hepatocellular carcinoma (HCC) cells under glucose deprivation conditions and in macrophages after extended LPS stimulation .

How can HRP-conjugated SESN2 antibodies be utilized to study SESN2's role in stress granule formation?

SESN2 has been implicated in stress granule dynamics, particularly in relation to stabilizing hexokinase 2 (HK2) mRNA. To study this role:

• Co-localization studies:

  • Use HRP-conjugated SESN2 antibodies alongside fluorescently-labeled stress granule markers (e.g., G3BP1)

  • Perform proximity ligation assays to visualize spatial relationships between SESN2 and stress granule components

• RNA-protein interaction analysis:

  • Combine RNA pull-down assays with SESN2 antibody detection to identify SESN2-RNA complexes

  • According to research, SESN2 competes with IGF2BP3 for binding to the 3'-UTR region of HK2 mRNA, affecting its stability

• Stress-induced dynamics:

  • Track SESN2 translocation to stress granules under various stressors (oxidative, ER stress, nutrient deprivation)

  • Time-course experiments have shown SESN2 association with mitochondria after 12h of LPS priming followed by ATP treatment

These approaches can reveal how SESN2 contributes to stress granule formation and the fate of specific mRNAs during cellular stress responses.

What are the best practices for using SESN2 antibodies in ChIP experiments to study epigenetic regulation?

When using SESN2 antibodies for chromatin immunoprecipitation (ChIP) studies:

• Crosslinking optimization:

  • For SESN2-associated chromatin studies, use 1% formaldehyde for 10 minutes at room temperature

  • Double crosslinking with disuccinimidyl glutarate (DSG) followed by formaldehyde can improve detection of indirect DNA associations

• Sonication parameters:

  • Optimize to achieve 200-500 bp DNA fragments

  • Verify fragment size by agarose gel electrophoresis

• Antibody selection:

  • For studying SESN2 regulation, combine SESN2 antibodies with antibodies against histone marks like H3K9me3 and H3K9ac

  • Research has shown SETDB1 regulates SESN2 expression by promoting H3K9me3 modifications at the SESN2 promoter

• Controls:

  • Include IgG control, input control, and positive control regions (known SESN2-regulated genes)

  • Use GAPDH as a negative control region, as studies show no significant accumulation of histone modifications in this region

• Data analysis:

  • Normalize to input and IgG controls

  • Compare enrichment across different experimental conditions

This approach has revealed that SETDB1 accumulates on the SESN2 promoter and promotes recruitment of H3K9me3, leading to transcriptional repression .

How should researchers interpret multiple SESN2 bands or inconsistent molecular weights in Western blots?

When encountering multiple bands or unexpected molecular weights:

• Post-translational modifications: SESN2 can undergo phosphorylation and ubiquitination , potentially altering migration patterns

• Isoform detection: Verify which isoforms your antibody recognizes and whether tissue-specific variants exist

• Degradation products: Fresh sample preparation and inclusion of protease inhibitors can minimize degradation

• Cross-reactivity: Test specificity using:

  • siRNA/shRNA knockdown: Knockdown experiments using shSESN2 (sequence: 5′-GGTCCACGTGAACTTGCTGC-3′) have successfully validated SESN2 bands

  • Knockout controls: Compare with Sesn2^-/- samples when available

• Stress-induced modifications: Under certain stress conditions, SESN2 can form complexes that alter its migration pattern

  • Research shows stress can induce SESN2-SQSTM1 interactions in both cytoplasmic and mitochondrial compartments

To distinguish between specific and non-specific bands, researchers should compare results across multiple antibodies and validate with genetic approaches.

How can SESN2 antibodies be utilized to study its role in mitophagy and mitochondrial quality control?

For investigating SESN2's role in mitophagy:

• Mitochondrial fractionation studies:

  • Use HRP-conjugated SESN2 antibodies to detect SESN2 recruitment to mitochondria

  • Research shows SESN2 associates with mitochondria after stress exposure

  • Include mitochondrial markers (e.g., TOM20) to confirm fractionation quality

• Co-immunoprecipitation approaches:

  • Immunoprecipitate with SESN2 antibodies to identify mitophagy-related binding partners

  • Studies have revealed SESN2 interactions with SQSTM1 in both cytoplasm and mitochondrial fractions after stimulation

• Functional assays:

  • Compare mitochondrial clustering and perinuclear aggregation between wild-type and Sesn2^-/- cells

  • Research indicates SESN2 facilitates mitochondrial priming by mediating SQSTM1 aggregation and binding to ubiquitinated mitochondria

• Mitophagy flux assessment:

  • Use SESN2 antibodies alongside mitophagy markers (LC3, PINK1, Parkin)

  • Sesn2^-/- mice display defective mitophagy upon immune stimulation

These approaches can help elucidate how SESN2 contributes to mitochondrial quality control, particularly in contexts like inflammation where SESN2-mediated mitophagy protects against NLRP3 inflammasome hyperactivation .

What are the optimal approaches for studying SESN2's role in IgE class switching in B cells?

When investigating SESN2's role in B cell immunology:

• Detection of SESN2 in B cells:

  • RT-PCR has confirmed Sesn2 mRNA expression in mouse B cell lines (L10A6.2 and A20.3) and purified splenic B cells

  • LPS stimulation enhances SESN2 expression in B cells

• Analyzing class switch recombination:

  • Use germline transcript (GLT) analysis:

    • IL-4-induced GLTε expression is reduced in Sesn2^-/- splenocytes

    • IL-4-induced GLTγ1 is also reduced in Sesn2^-/- cells

    • Other cytokine-induced GLTs (GLTγ3, GLTγ2b, GLTα, GLTγ2c) are not affected by SESN2 deficiency

• Promoter activity assays:

  • Luciferase reporter assays have shown that SESN2 overexpression enhances IL-4-induced GLTε promoter activity

  • SESN2 affects IgE CSR by enhancing IL-4-induced GLTε transcription

• AMPK pathway investigation:

  • AMPK activation (phosphorylated AMPK; p-AMPK) is reduced in Sesn2^-/- B cells

  • AICAR (an AMPK activator) rescues reduced IL-4-induced sIgE expression in Sesn2^-/- B cells