rabbit monoclonal anti nrf2 d1z9c  (Cell Signaling Technology Inc)

Journal: Redox Biology

Article Title: Considerations for antibody-based detection of NRF2 in human cells

doi: 10.1016/j.redox.2025.103549

Figure Lengend Snippet: NRF2 antibodies targeting different epitopes detect 3 bands migrating between 100 and 130 kDa in 8% Tris-glycine SDS-PAGE. (A) NRF2 migration in Tris-glycine 8 % SDS-PAGE in H1299, RERF and A549 cells at steady state and upon treatment with tert-butylhydroquinone (t-BHQ). (B) Knockdown of NRF2 gene ( NFE2L2 ) with a pool of siRNA targeting NFE2L2 (siNRF2) compared to transfection with control unspecific short RNAs (ctrl) in H1299 and RERF cells. (C,D) NRF2 dephosphorylation with λ phosphatase in RERF and H1299 cell lysates. Lysates were incubated with or without λ phosphatase for 30 min in 30 °C in the presence of MnCl 2 and NRF2 was detected by western blot. Arrows indicate bands detected by anti-NRF2 antibodies from Abcam [EP1808Y] (C) and Cell Signaling [D1Z9C] (D). (E) Detection of NRF2 in cytoplasmic and nuclear fractions upon treatment with tert-BHQ in H1299, RERF and A549 cells. Arrows indicate signal detected by anti-NRF2 antibodies from Abcam [EP1808Y]. Alfa-tubulin is a marker of cytoplasmic fraction and lamin is a marker of nuclear fraction. (F) Pulse-chase experiment upon translation inhibition with emetine in RERF cells. Cells were treated with emetine for indicated time and NRF2 levels were detected with Abcam [EP1808Y] antibodies. Molecular Weight is depicted at each western blot.

Article Snippet: Only one monoclonal anti-NRF2 antibody that we analyzed, Cell Signaling clone E5F1A, did not bind to calmegin in H1299 cells or showed little binding in RERF cells ( C).

Techniques: SDS Page, Migration, Knockdown, Transfection, Control, De-Phosphorylation Assay, Incubation, Western Blot, Marker, Pulse Chase, Inhibition, Molecular Weight

Journal: Redox Biology

Article Title: Considerations for antibody-based detection of NRF2 in human cells

doi: 10.1016/j.redox.2025.103549

Figure Lengend Snippet: Calmegin is precipitated by anti-NRF2 antibodies and co-migrates with NRF2 in SDS-PAGE. (A) A scheme representing the workflow of identification of proteins precipitated by anti-NRF2 antibodies in RERF cells were treated with translation inhibitor emetine for 2 h cells or with tert-BHQ for 5 h. NRF2 was precipitated with Abcam EP1808Y antibodies. Precipitates were resolved in 8 % Tris-glycine SDS-PAGE and stained with Flamingo Fluorescent Gel Stain. Gel pieces with proteins migrating in 100–130 kDa range, representing NRF2 signal in western blot, were excised, followed by in-gel tryptic digestion of proteins. Diagrams show peptides of NRF2 (A) and calmegin (B) detected with LC-MS/MS in each sample (in duplicates). No NRF2 peptides were detected upon translation inhibition with emetine.

Article Snippet: Only one monoclonal anti-NRF2 antibody that we analyzed, Cell Signaling clone E5F1A, did not bind to calmegin in H1299 cells or showed little binding in RERF cells ( C).

Techniques: SDS Page, Staining, Western Blot, Liquid Chromatography with Mass Spectroscopy, Inhibition

Journal: Redox Biology

Article Title: Considerations for antibody-based detection of NRF2 in human cells

doi: 10.1016/j.redox.2025.103549

Figure Lengend Snippet: Various commercial monoclonal anti-NRF2 antibodies bind calmegin. Upon calmegin (CLGN) knockdown in RERF (A) and H1299 (B) cells, NRF2 was detected with three different monoclonal anti-NRF2 antibodies: Abcam EP1808Y, Cell Signaling D1Z9C and ABclonal ARC0806. All these three antibodies bind to calmegin. (C) Anti-NRF2 antibody from Cell Signaling (E5F1A) binds little calmegin in RERF or no detectable calmegin in H1299 cells. (D) RERF cells were knocked down for NRF2 expression (siNRF2) or transfected with control short RNA (Ctrl) for 48 h and treated with NRF2 activator tert-BHQ (tBHQ). NRF2 was detected with anti-NRF2 antibodies from Cell Signaling (E5F1A).

Article Snippet: Only one monoclonal anti-NRF2 antibody that we analyzed, Cell Signaling clone E5F1A, did not bind to calmegin in H1299 cells or showed little binding in RERF cells ( C).

Techniques: Knockdown, Expressing, Transfection, Control

Journal: Redox Biology

Article Title: Considerations for antibody-based detection of NRF2 in human cells

doi: 10.1016/j.redox.2025.103549

Figure Lengend Snippet: NRF2 domain structure with marked localization of immunogens used for the production of monoclonal antibodies used in this study, marked by a wave (∼), based on the information provided by a manufacturer. Antibodies which bind NRF2 with the highest specificity (Cell Signaling E5F1A) are marked with blue. Cell Signaling E5F1A - immunogen is a synthetic peptide corresponding to residues surrounding Ile86 ; Cell Signaling D1Z9C - immunogen is a synthetic peptide corresponding to residues surrounding Ala275 ; Abcam EP1808Y - immunogen is a synthetic peptide surrounding Leu550 ; ABclonal A3577 - immunogen is a synthetic peptide corresponding to a sequence within amino acids 505–605.

Article Snippet: Only one monoclonal anti-NRF2 antibody that we analyzed, Cell Signaling clone E5F1A, did not bind to calmegin in H1299 cells or showed little binding in RERF cells ( C).

Techniques: Bioprocessing, Sequencing

Journal: Redox Biology

Article Title: Considerations for antibody-based detection of NRF2 in human cells

doi: 10.1016/j.redox.2025.103549

Figure Lengend Snippet: Analysis of selected monoclonal anti-NRF2 antibodies specificity in immunofluorescence in H1299 cells. Cells were knocked down for NRF2 expression (siNRF2) or transfected with control short RNA (Ctrl) for 48 h and, where indicated, treated with tert-BHQ (tBHQ) for 5 h. After that cells were fixed with 4 % para-formaldehyde and permabilized with 0.2 % Triton X-100, followed by staining with various anti-NRF2 antibodies (A,D,E) or analyzed for NRF2 levels in western blot (B). Alternatively, non-transfected cells were stained with anti-calmegin (CLGN) antibodies (C). Nuclei were stained with DAPI. All experiments were performed in at least three independent biological repeats with two technical repeats for each condition.

Article Snippet: Only one monoclonal anti-NRF2 antibody that we analyzed, Cell Signaling clone E5F1A, did not bind to calmegin in H1299 cells or showed little binding in RERF cells ( C).

Techniques: Immunofluorescence, Expressing, Transfection, Control, Staining, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Formononetin promotes porcine oocytes maturation and improves embryonic development by reducing oxidative stress

doi: 10.3389/fcell.2024.1520429

Figure Lengend Snippet: Information about the primer sequences.

Article Snippet: And incubation with horseradish peroxidase-conjugated secondary antibodies at RT for 1 h. Primary antibodies were rabbit monoclonal antibodies against NRF2 (1:2000, #16396-1-AP, Proteintech), KEAP1 (1:2000, #10503-2-AP, Proteintech), and β-Actin (1:5000, #20536-1-AP, Proteintech).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Formononetin promotes porcine oocytes maturation and improves embryonic development by reducing oxidative stress

doi: 10.3389/fcell.2024.1520429

Figure Lengend Snippet: Effect of FMN on mitochondrial function in oocytes and early embryos. (A) The mitochondria distribution at MII stage were stained with Mito-Tracker Red; scale bar, 50 μm. (B) The relative abundance of mitochondria in oocyte were analyzed for control and FMN-treated group. (C) Relative mRNA levels of ATP5B, NRF2 and KEAP1 in oocytes. (D) 4-cell stage embryos stained with JC-1. Scale bar, 100 μm. (E) Relative levels of JC-1 Red/Green fluorescence intensity in embryos were analyzed for control and FMN-treated group. (F) Relative mRNA levels of TFAM and NRF1 in embryos. (G) ATP levels of 4-cell stage embryos in both control and FMN-treated groups. Data are presented as the mean ± standard deviation (SD). * p < 0.05,** p < 0.01, *** p < 0.001 vs. 0 μM FMN group.

Article Snippet: And incubation with horseradish peroxidase-conjugated secondary antibodies at RT for 1 h. Primary antibodies were rabbit monoclonal antibodies against NRF2 (1:2000, #16396-1-AP, Proteintech), KEAP1 (1:2000, #10503-2-AP, Proteintech), and β-Actin (1:5000, #20536-1-AP, Proteintech).

Techniques: Staining, Control, Fluorescence, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Formononetin promotes porcine oocytes maturation and improves embryonic development by reducing oxidative stress

doi: 10.3389/fcell.2024.1520429

Figure Lengend Snippet: Effect of FMN on the expression levels of the Nrf2/Keap1 signaling pathway-related protein and genes in porcine blastocysts. (A) Porcine blastocysts incubated with or without FMN were stained with NRF2 (green) and DAPI (blue), scale bar, 50 μm. (B) Relative levels of NRF2 fluorescence intensity in blastocysts were analyzed for the control and FMN-treated group. (C) Relative mRNA expression levels of genes related to Nrf2/Keap1 pathway, NRF2, KEAP1, NQO1, UCHL1 in blastocysts. (D) Western blot analysis of NRF2 and KEAP1 protein expressions in blastocysts in the control and FMN-treated groups. Data were presented as the mean ± standard deviation (SD). ** p < 0.01,*** p < 0.001 vs. 0 μM FMN group.

Article Snippet: And incubation with horseradish peroxidase-conjugated secondary antibodies at RT for 1 h. Primary antibodies were rabbit monoclonal antibodies against NRF2 (1:2000, #16396-1-AP, Proteintech), KEAP1 (1:2000, #10503-2-AP, Proteintech), and β-Actin (1:5000, #20536-1-AP, Proteintech).

Techniques: Expressing, Incubation, Staining, Fluorescence, Control, Western Blot, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Formononetin promotes porcine oocytes maturation and improves embryonic development by reducing oxidative stress

doi: 10.3389/fcell.2024.1520429

Figure Lengend Snippet: Summary of the effect of Formononetin (FMN) on porcine oocyte in vitro maturation and early embryo in vitro . This schematic view illustrates that during the IVM stage, FMN enhances oocyte maturation in vitro by up-regulating genes related to CCs expansion and antioxidants, reducing oxidative stress, and improving mitochondrial function. During the IVC stage, FMN promotes the nuclear expression of Nrf2 through the P62-Keap1-Nrf2 pathway, which exerts antioxidant function and enhances mitochondrial function. Then, FMN promotes cell proliferation, reduces apoptosis, and lowers autophagy levels.

Article Snippet: And incubation with horseradish peroxidase-conjugated secondary antibodies at RT for 1 h. Primary antibodies were rabbit monoclonal antibodies against NRF2 (1:2000, #16396-1-AP, Proteintech), KEAP1 (1:2000, #10503-2-AP, Proteintech), and β-Actin (1:5000, #20536-1-AP, Proteintech).

Techniques: In Vitro, Expressing

Journal: International journal of molecular sciences

Article Title: Potential of NRF2 Inhibitors-Retinoic Acid, K67, and ML-385-In Overcoming Doxorubicin Resistance in Promyelocytic Leukemia Cells.

doi: 10.3390/ijms251910257

Figure Lengend Snippet: Figure 1. Chemical structures of NRF2 inhibitors.

Article Snippet: The membranes were blocked with 5% milk in 0.1% Tween-20/TBS (Tris-buffered saline) for 1 h at room temperature and they were incubated with rabbit monoclonal anti-NRF2 antibody (#12721, Cell Signaling Technology, Danvers, MA, USA), diluted 1:1000 in 5% milk in 0.1% TBST or anti-KEAP1 (#8047, Cell Signaling Technology, Danvers, MA, USA), diluted 1:1000 in 5% bovine serum albumin (BSA) in 0.1% TBST overnight at 4 ◦C.

Techniques:

Journal: International journal of molecular sciences

Article Title: Potential of NRF2 Inhibitors-Retinoic Acid, K67, and ML-385-In Overcoming Doxorubicin Resistance in Promyelocytic Leukemia Cells.

doi: 10.3390/ijms251910257

Figure Lengend Snippet: Figure 8. Protein expression of NRF2 (#12721, Cell Signaling Technology, Danvers, MA, USA) (A,C) and KEAP1 (#8047, Cell Signaling Technology, Danvers, MA, USA) (B,C) in HL-60/DR cells pre- incubated with ML-385 for 24 h and treated with 100 nM doxorubicin (DOXO) for a subsequent 24 h was visualized by Western blot. The intensity of bands corresponding to proteins was analyzed by densitometry. The results are shown as the fold change of proteins levels of treated cells vs. control HL-60/DR cells. β-actin (sc-477778, Santa Cruz Biotechnology, Santa Cruz, CA, USA) served as the loading control. The figure shows mean results ± SD, n = 4, *** p < 0.001 compared to control without doxorubicin and ML-385, and # p < 0.05 compared to control with doxorubicin and without ML-385. Statistical analysis was conducted using the U Mann–Whitney test.

Article Snippet: The membranes were blocked with 5% milk in 0.1% Tween-20/TBS (Tris-buffered saline) for 1 h at room temperature and they were incubated with rabbit monoclonal anti-NRF2 antibody (#12721, Cell Signaling Technology, Danvers, MA, USA), diluted 1:1000 in 5% milk in 0.1% TBST or anti-KEAP1 (#8047, Cell Signaling Technology, Danvers, MA, USA), diluted 1:1000 in 5% bovine serum albumin (BSA) in 0.1% TBST overnight at 4 ◦C.

Techniques: Expressing, Incubation, Western Blot, Control, MANN-WHITNEY

Journal: International journal of molecular sciences

Article Title: Potential of NRF2 Inhibitors-Retinoic Acid, K67, and ML-385-In Overcoming Doxorubicin Resistance in Promyelocytic Leukemia Cells.

doi: 10.3390/ijms251910257

Figure Lengend Snippet: Figure 9. Experimental schemes of HL-60 and HL-60/DR cells. (1) HL-60 cells and (2) HL-60/DR cells were incubated with doxorubicin. (3) HL-60/DR cells were pre-incubated with NRF2 inhibitors followed by doxorubicin treatment.

Article Snippet: The membranes were blocked with 5% milk in 0.1% Tween-20/TBS (Tris-buffered saline) for 1 h at room temperature and they were incubated with rabbit monoclonal anti-NRF2 antibody (#12721, Cell Signaling Technology, Danvers, MA, USA), diluted 1:1000 in 5% milk in 0.1% TBST or anti-KEAP1 (#8047, Cell Signaling Technology, Danvers, MA, USA), diluted 1:1000 in 5% bovine serum albumin (BSA) in 0.1% TBST overnight at 4 ◦C.

Techniques: Incubation