Phospho-Nrf2 (Ser40) Antibody (YA168)

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.

Western blot analysis of extracts from HepG2 (lane 1(20μg)) 、Raji (lane 2(20μg)) 、HeLa (lane 3(20μg)) 、Mouse liver (lane 4(20μg)) 、Rat liver (lane 5(20μg)) 、Mouse lung (lane 6(20μg)) and Rat lung (lane 7(20μg)) using Phospho-Nrf2 (Ser40) Antibody (HY-P80471) . Proteins were transferred to a PVDF membrane and blocked with 5% BSA in TBST for 1.5 hour at room temperature. The primary antibody (1/1000) and Loading control antibody (Hsp90, 1/10000) was used in 5% BSA in TBST at 4℃ overnight. Goat Anti-Rabbit IgG-HRP Secondary Antibody (1/10,000) was used for 1 hour at room temperature.

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).

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).