Phospho-Nrf2 (Ser40) Antibody (YA168)

Phospho-Nrf2 (Ser40) Antibody (YA168) is a Rabbit-derived and non-conjugated IgG monoclonal antibody, targeting to Phospho-Nrf2 (Ser40).

NFE2L2, a pivotal transcription factor, assumes a central role in the cellular response to oxidative stress. It binds to antioxidant response elements (ARE) in the promoter regions of numerous cytoprotective genes, including phase 2 detoxifying enzymes, promoting their expression and neutralizing reactive electrophiles. Under normal conditions, NFE2L2 is ubiquitinated and degraded in the cytoplasm by the BCR(KEAP1) complex. However, in the face of oxidative stress, electrophile metabolites inhibit the BCR(KEAP1) complex, leading to nuclear accumulation of NFE2L2, heterodimerization with small Maf proteins, and binding to ARE elements in cytoprotective target genes. The NFE2L2/NRF2 pathway is also activated in response to selective autophagy, where autophagy facilitates the interaction between KEAP1 and SQSTM1/p62, inactivating the BCR(KEAP1) complex and allowing for NFE2L2 nuclear accumulation. Apart from its role in oxidative stress response, NFE2L2 contributes to the transcriptional activation of genes in the beta-globin cluster and plays a critical role in regulating the innate immune response and antiviral cytosolic DNA sensing. It acts as a key regulator during sepsis, maintaining redox homeostasis and restraining dysregulation in pro-inflammatory signaling pathways. Furthermore, NFE2L2 suppresses the macrophage inflammatory response, inhibiting pro-inflammatory cytokine transcription and the induction of IL6. It also represses antiviral cytosolic DNA sensing by suppressing the expression of the adapter protein STING1, limiting the release of pro-inflammatory cytokines in response to human coronavirus SARS-CoV-2 infection and other pathogenic viruses. Activation of NFE2L2 is facilitated by cell-derived metabolites, including itaconate and fumarate.

Free

Q16236

Predicted band size: 68 kDa;Observed band size: 100 kDa

Protein A affinity purified.

Cytoplasm, Nucleus.

Non-conjugated

Phosphorylated

AB_3102545

Epigenetics and Nuclear Signaling

Primary Antibody; Recombinant Rabbit Monoclonal Antibody

Recombinant, Monoclonal

Rabbit

Human

WB: 1:1000-1:5000; ICC/IF: 1:50-1:200; IHC-P: 1:50-1:200; FC: 1:50-1:100; IP: Use at an assay dependent concentration.

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  Western blot analysis of extracts from HepG2 (lane 2(20μg), Raji (lane 3(20μg) and HEK293(lane 4(20μg) using Phospho-Nrf2 (Ser40) (HY-P80471) Rabbit mAb. Proteins were transferred to a PVDF membrane and blocked with 5% non-fat milk in TBST for 2 hour at room temperature. The primary antibody (1/1000) and Loading control antibody (Beta Actin, HY-P80438, 1/10000) was used in 5% non-fat milk in TBST at 4°C overnight. Goat Anti-Mouse/Rabbit IgG-HRP Secondary Antibody (1/10000) was used for 1 hour at room temperature.
• 
  Immunocytochemistry analysis of A549 cells with 2μg/mL LPS for 4 hours labeling Phospho-Nrf2 (Ser40) with Phospho-Nrf2 (Ser40) antibody (HY-P80471) at 1/50 dilution. Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature, then blocked with QuickBlock™ Blocking Buffer for Immunol Staining for 10 min at room temperature. Cells were then incubated with Phospho-Nrf2 (Ser40) antibody (HY-P80471) at 1/50 dilution in QuickBlock™ Blocking Buffer for Immunol Staining at 4℃. Alexa Fluor® 488-conjugated AffiniPure Goat Anti-Rabbit IgG H&L（HY-P8002, Green) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. The Nuclear counterstain was DAPI (Blue).
• 
  Immunocytochemistry analysis of HepG2 cells labeling Phospho-Nrf2 (Ser40) with Phospho-Nrf2 (Ser40) antibody (HY-P80471) at 1/50 dilution. Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature, then blocked with QuickBlock™ Blocking Buffer for Immunol Staining for 10 min at room temperature. Cells were then incubated with Phospho-Nrf2 (Ser40) antibody (HY-P80471) at 1/50 dilution in QuickBlock™ Blocking Buffer for Immunol Staining at 4℃. Alexa Fluor® 488-conjugated AffiniPure Goat Anti-Rabbit IgG H&L（HY-P8002, Green) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. The Nuclear counterstain was DAPI (Blue).
• 
  Immunohistochemical analysis of paraffin-embedded Mouse liver tissue using Phospho-Nrf2 (Ser40) Antibody (YA168). The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 8 minutes. The tissues were blocked in QuickBlock for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HY-P80471, 1/1000) in 4℃ overnight. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
• 
  Immunohistochemical analysis of paraffin-embedded Mouse liver tissue using Phospho-Nrf2 (Ser40) Antibody (YA168). The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 8 minutes. The tissues were blocked in QuickBlock for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HY-P80471, 1/1000) in 4℃ overnight. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
• 
  Flow cytometric analysis of 1X10^6 K562 cells labeling Phospho-Nrf2 (Ser40) Antibody (HY-P80471, red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Then stained with the primary antibody at 1/50 dilution for an hour at 4℃. Alexa Fluor® 488-conjugated AffiniPure Goat Anti-Rabbit IgG H&L (HY-P8002) was used as the secondary antibody at 1/1,000 dilution for 30 minutes at 4℃. Rabbit IgG Isotype Control (HY-P80879, blue) was used as the isotype control, cells without incubation with primary antibody were used as the unlabeled control (black).

WB, ICC/IF, IHC-P, FC, IP

Liquid

Supplied in 1*TBS (pH7.4), 0.05% BSA and 40% Glycerol. Preservative: 0.05% Sodium Azide.

Stored at -20°C for 1 year. Avoid repeated freeze / thaw cycles.

Shipping with blue ice.

IgG

Endogenous

Synthetic phosphopeptide corresponding to residues surrounding Ser40 of Human Nrf2.AA range:20-69.