nrf2 (d1z9c) xp rabbit mab antibody  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: NRF2 regulates lipid droplet dynamics to prevent lipotoxicity

doi: 10.1016/j.isci.2025.112925

Figure Lengend Snippet: NRF2 stability and activity influences LD accumulation in HEK-TtH cells (A) Immunoblot confirming NRF2 activation in sgKEAP1 cells. (B) Quantification of LD area per cell from (C). n = 4 biological replicates. Mean ± SD. (C) Representative images of LDs in sgKEAP1 HEK-TtH cells taken with 63× objective. Scale bars represent 20 μm. (D) Immunoblot confirming NRF2 activation in cells stimulated with 10 μM Sulforaphane for 24 h. (E) Quantification of LD area per cell from (F). n = 4 biological replicates. Mean ± SD. (F) Representative images of LDs in HEK-TtH cells treated with 10 μM sulforaphane for 24 h taken with 63× objective. Scale bars represent 20 μm. (G) Immunoblot confirming NRF2 depletion in sgNRF2 cells. (H) Quantification of LD area per cell from (I). n = 3 biological replicates. Mean ± SD. (I) Representative images of LDs in sgNRF2 HEK-TtH cells taken with 63× objective. Scale bars represent 20 μm. (J) Immunoblot confirming NRF2 depletion and inactivation of transcriptional activity in sgKEAP1 cells with addition of sgNRF2. (K) Quantification of LD area per cell from (L). n = 3 biological replicates. Mean ± SD. (L) Representative images of LDs in sgKEAP1/sgNRF2 double-KO HEK-TtH cells taken with 63× objective. Scale bars represent 20 μm.

Article Snippet: NRF2 (D1Z9C) XP Rabbit mAB , Cell Signaling Technology , Cat# 12721; RRID: AB_2715528.

Techniques: Activity Assay, Western Blot, Activation Assay

Journal: iScience

Article Title: NRF2 regulates lipid droplet dynamics to prevent lipotoxicity

doi: 10.1016/j.isci.2025.112925

Figure Lengend Snippet: NRF2-mediated LD accumulation occurs in specific lung cancer cell lines (A) Representative images of LDs in lung cancer cells taken with 63× objective. Scale bars represent 20 μm. (B) Quantification of LD area per cell from (A). n = 3 biological replicates. Mean ± SD. (C) Immunoblot of basal NRF2 and HMOX1 levels in lung cancer cell lines. (D) Relative basal mRNA expression (2-ΔΔCT relative to GAPDH) of NRF2 transcriptional targets in lung cancer cell lines. Data normalized to H1650 mRNA expression. Horizontal dotted line represents no change relative to H1650 mRNA levels. n ≥ 3 biological replicates. Mean ± SD. (E) Immunoblot confirming NRF2 depletion in sgNRF2 H1650 cells. (F) Immunoblot confirming NRF2 depletion in sgNRF2 H2009 cells. (G) Representative images of LDs in sgNRF2 H1650 cells taken with 63× objective. Scale bars represent 20 μm. (H) Representative images of LDs in sgNRF2 H2009 cells taken with 63× objective. Scale bars represent 20 μm. (I) Quantification of LD area per cell from (G). n = 3 biological replicates. Statistics represented calculated by Student’s t test (one-tailed, unpaired with equal variance). Mean ± SD. (J) Quantification of LD area per cell from (G). Horizontal dotted line represents no change relative to sgScr cells. n = 3 biological replicates. Statistics represented calculated by Student’s t test (one-tailed, and paired). Mean ± SD. (K) Quantification of LD area per cell from (H). n = 3 biological replicates. Statistics represented calculated by Student’s t test (one-tailed, unpaired with equal variance). Mean ± SD. (L) Quantification of LD area per cell from (H). Horizontal dotted line represents no change relative to sgScr cells. n = 3 biological replicates. Statistics represented calculated by Student’s t test (one-tailed, and paired). Mean ± SD.

Article Snippet: NRF2 (D1Z9C) XP Rabbit mAB , Cell Signaling Technology , Cat# 12721; RRID: AB_2715528.

Techniques: Western Blot, Expressing, One-tailed Test

Journal: iScience

Article Title: NRF2 regulates lipid droplet dynamics to prevent lipotoxicity

doi: 10.1016/j.isci.2025.112925

Figure Lengend Snippet: The negative regulator of adipose triglyceride lipase, G0S2, acts downstream of NRF2 to regulate LD abundance in HEK-TtH cells (A) Fold change of mRNA expression (2-ΔΔCT relative to GAPDH) of regulators of LD structure, lipids, and lipid storage in sgKEAP1 cells vs. sgScr cells. Vertical dotted line represents no change relative to sgScr control mRNA levels. n = 3 biological replicates. Mean ± SD. ( p < 0.05 = ∗, p < 0.01 = ∗∗). (B) Fold change of mRNA expression (2-ΔΔCT relative to GAPDH) of G0S2 in lung cancer cell lines. Data represented normalized to G0S2 expression of H1650 cells. Horizontal dotted line represents no change relative to H1650 mRNA levels. n = 3 biological replicates. Mean ± SD. (C) Representative images of LDs in sgKEAP1/sgG0S2 double-KO HEK-TtH cells taken with 63× objective. Scale bars represent 20 μm. (D) Quantification of LD area per cell from (C). n = 5 biological replicates. Statistics represented calculated by Student’s t test (one-tailed, unpaired with equal variance). Mean ± SD. (E) Representative images of LDs in sgNRF2/mG0S2-Flag-overexpression HEK-TtH cells taken with 63× objective. Scale bars represent 20 μm. (F) Quantification of LD area per cell from (E). n = 3 biological replicates. Statistics represented calculated by Student’s t test (one-tailed, unpaired with equal variance). Mean ± SD.

Article Snippet: NRF2 (D1Z9C) XP Rabbit mAB , Cell Signaling Technology , Cat# 12721; RRID: AB_2715528.

Techniques: Expressing, Control, One-tailed Test, Over Expression

Journal: iScience

Article Title: NRF2 regulates lipid droplet dynamics to prevent lipotoxicity

doi: 10.1016/j.isci.2025.112925

Figure Lengend Snippet: Specific fatty acids stabilize NRF2 and drive downstream signaling to protect cells from lipotoxic stress (A) Representative images of LDs in HEK-TtH cells treated with fatty acids conjugated to fatty acid-free BSA for 4 h taken with 63× objective. Scale bars represent 20 μm. (B) Quantification of LD area per cell from (A). n = 3 independent biological replicates. Mean ± SD. ( p < 0.05 = ∗, p < 0.01 = ∗∗, p < 0.001 = ∗∗∗). (C) Quantification of cell viability based on luminescence from CellTiter-Glo Luminescent Cell Viability Assay for HEK-TtH cell treated with gradient of fatty acids conjugated to fatty acid-free BSA relative to vehicle controls for 24 h. Horizontal dotted line represents half maximal inhibitory concentration (IC50). n = 3 biological replicates of each. (D) IC50 and standard error calculated from (C) for each fatty acid treatment. (E) Immunoblot of NRF2 and downstream transcriptional target HMOX1 after 4 h of treatment of fatty acids conjugated to fatty acid-free BSA. (F) Quantification of cell viability based on luminescence from CellTiter-Glo Luminescent Cell Viability Assay for HEK-TtH sgNRF2 and sgKEAP1 cell lines treated with 200 μM arachidonic acid (AA) conjugated to fatty acid-free BSA for 24 h. n = 3 biological replicates of each. Mean ± SD. (G) Quantification of cell viability based on luminescence from CellTiter-Glo Luminescent Cell Viability Assay for H2009 sgNRF2 lung cancer cell lines treated with 200 μM arachidonic acid (AA) conjugated to fatty acid-free BSA for 24 h. n = 3 biological replicates of each. Mean ± SD. (H) Immunoblot comparing 4-hydroxynonenal in sgScr and sgNRF2 cells after 4 h treatment with gradient of arachidonic acid conjugated to fatty acid-free BSA. (I) Quantification of cell viability based on luminescence from CellTiter-Glo Luminescent Cell Viability Assay of WT HEK-TtH cells treated with 200 μM arachidonic acid (AA) conjugated to fatty acid-free BSA, co-treated with 10 μM Ferrostatin-1 (Fer-1) or 10 μM Z-VAK-FMK for 24 h. n = 3 biological replicates of each. Mean ± SD.

Article Snippet: NRF2 (D1Z9C) XP Rabbit mAB , Cell Signaling Technology , Cat# 12721; RRID: AB_2715528.

Techniques: Cell Viability Assay, Concentration Assay, Western Blot

Journal: iScience

Article Title: NRF2 regulates lipid droplet dynamics to prevent lipotoxicity

doi: 10.1016/j.isci.2025.112925

Figure Lengend Snippet: Inhibition of lipid droplet degradation protects cells from ferroptosis (A) Immunoblot of NRF2 stability and downstream transcriptional target HMOX1 after treatment with 200 nM RSL3 for 4 h. (B) Quantification of cell viability based on luminescence from CellTiter-Glo Luminescent Cell Viability Assay for HEK-TtH cell treated with gradient of RSL3 relative to DMSO controls for 24 h. Horizontal dotted line represents half maximal inhibitory concentration (IC50). n = 4 biological replicates of each. (C) IC50 and standard error calculated from (B) for each lung cancer cell line treated with RSL3 gradient. (D) Quantification of luminescence from CellTiter-Glo Luminescent Cell Viability Assay of sgNRF2 H2009 cell lines treated with 200 nM RSL3 relative to vehicle control for 24 h. n = 3 biological replicates of each. Mean ± SD. (E) Quantification of luminescence from CellTiter-Glo Luminescent Cell Viability Assay of sgNRF2 and sgKEAP1 HEK-TtH cell lines treated with 200 nM RSL3 relative to vehicle control for 24 h. n = 6 biological replicates of each. Mean ± SD. (F) Representative images of LDs of HEK-TtH cells treated with 50 nM RSL3 for 24 h taken with 63× objective. Scale bars represent 20 μm. (G) Quantification of LD area per cell from (F). n = 3 biological replicates. Mean ± SD. (H) Quantification of cell viability from CellTiter-Glo Glo Luminescent Cell Viability Assay of sgNRF2 HEK-TtH cells treated with DMSO or 10 μM ATGListatin (ATGLi) in combination with 25 nM RSL3. n = 4 biological replicates. Statistics represented calculated by Student’s t test (one-tailed, unpaired with equal variance). Mean ± SD. (I) Representation of data from (H) when paired within each individual biological replicate. Statistics represented calculated by Student’s t test (one-tailed, paired). Mean ± SD. (J) Quantification of cell viability from CellTiter-Glo Glo Luminescent Cell Viability Assay of sgNRF2 HEK-TtH cells treated with DMSO or 10 μM ATGListatin (ATGLi) in combination with 50 nM RSL3. n = 4 biological replicates. Statistics represented calculated by Student’s t test (one-tailed, unpaired with equal variance). Mean ± SD. (K) Representation of data from (J) when paired within each individual biological replicate. Statistics represented calculated by Student’s t test (one-tailed, paired). Mean ± SD. (L) Quantification of cell viability from CellTiter-Glo Glo Luminescent Cell Viability Assay of sgNRF2 HEK-TtH cells overexpressing mG0S2-Flag or empty vector (EV) treated with 25 nM RSL3. n = 5 biological replicates. Statistics represented calculated by Student’s t test (one-tailed, unpaired with equal variance). Mean ± SD. (M) Representation of data from (L) when paired within each individual biological replicate. Statistics represented calculated by Student’s t test (one-tailed, paired). Mean ± SD. (N) Quantification of cell viability from CellTiter-Glo Glo Luminescent Cell Viability Assay of sgNRF2 HEK-TtH cells overexpressing mG0S2-Flag or empty vector (EV) treated with 50 nM RSL3. n = 5 biological replicates. Statistics represented calculated by Student’s t test (one-tailed, unpaired with equal variance). Mean ± SD. (O) Representation of data from (N) when paired within each individual biological replicate. Statistics represented calculated by Student’s t test (one-tailed, paired). Mean ± SD.

Article Snippet: NRF2 (D1Z9C) XP Rabbit mAB , Cell Signaling Technology , Cat# 12721; RRID: AB_2715528.

Techniques: Inhibition, Western Blot, Cell Viability Assay, Concentration Assay, Control, One-tailed Test, Plasmid Preparation

Journal: Molecules (Basel, Switzerland)

Article Title: Protective Role of Whey Protein Isolate on MPP + -Induced Differentiation of SH-SY5Y Cells by Modulating the Nrf2 Antioxidant Pathway.

doi: 10.3390/molecules30102207

Figure Lengend Snippet: Figure 3. WPI decreased the production of intracellular ROS in MPP+-treated differentiated SH-SY5Y cells through the accumulation of Nrf2 in the nucleus. (a) The assessment of intracellular relative ROS levels in differentiated SH-SY5Y cells exposed to MPP+ and WPI, with Hyd used as a positive control. (b) The examination of Nrf2 nuclear translocation in differentiated SH-SY5Y after co-treatment with WPI and MPP+. Nrf2 nuclear translocation was confirmed by immunofluorescence staining using Nrf2 antibody (green) and TOPRO-3 dye (red) (scale bar = 100 µm). Merged images displaying a yellow color (resulting from the merging of green and red) within the nucleus indicate the successful nuclear translocation of Nrf2. (c) The quantification of the nuclear–cytoplasmic signal ratio. Data are presented as mean ± SEM; * p ≤0.05 and *** p ≤0.001 vs. MPP+-treated group, # p ≤0.05 and ### p ≤0.001 vs. control group.

Article Snippet: The iScript Reverse Transcription Supermix and iTaq Universal SYBR Green Supermix were from Bio-Rad (Berkeley, CA, USA), and the NRF2 (D1Z9C) XP® Rabbit mAb was from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Positive Control, Translocation Assay, Immunofluorescence, Staining, Control

Journal: Molecules (Basel, Switzerland)

Article Title: Protective Role of Whey Protein Isolate on MPP + -Induced Differentiation of SH-SY5Y Cells by Modulating the Nrf2 Antioxidant Pathway.

doi: 10.3390/molecules30102207

Figure Lengend Snippet: Figure 5. The schematic of the proposed mechanism illustrates that WPI, at a concentration of 5 µg/mL, exerts antioxidant properties on differentiated SH-SY5Y cells. WPI may be internalized by differentiated SH-SY5Y cells and exert bioactivity. WPI induces the translocation of Nrf2 into the nucleus, resulting in the enhanced expression of antioxidants such as GPx and HO1. Following this, these antioxidants decrease intracellular ROS, a precursor to cell death. Excessive intracellular ROS is induced by mitochondrial dysfunction caused by MPP+, which enters differentiated SH-SY5Y cells through the dopamine transporter (DAT). GPx converts H2O2 to water molecules. HO1 changes heme, which is a toxic molecule, to biliverdin, which is subsequently converted to bilirubin, which is an antioxidant molecule that can convert hydroxyl radicals (•OH) to water molecules. The figures were produced through the utilization of the BioRender.com platform.

Article Snippet: The iScript Reverse Transcription Supermix and iTaq Universal SYBR Green Supermix were from Bio-Rad (Berkeley, CA, USA), and the NRF2 (D1Z9C) XP® Rabbit mAb was from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Concentration Assay, Translocation Assay, Expressing, Produced

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: The NFE2L2 (NRF2) transcription factor controls genes involved in the oxidative stress response and inflammation in myometrial cells.

doi: 10.1016/j.bbamcr.2025.119985

Figure Lengend Snippet: Fig. 2. Confirmation of NFE2L2 knockdown in PHM1–31 myometrial cells and measurement of ROS in NFE2L2 knockdown cells. PHM1–31 cells were transfected with siRNA (scrambled, si1, si2 or si3) and NFE2L2 knockdown analyzed in the presence or absence of IL1B (10 ng/ml). (A) Induction of NFE2L2 by IL1B treatment was measured in PHM1–31 cells in the presence and absence of IL1B (10 ng/ml) for 4 h using quantitative PCR (n = 4). (B) NFE2L2 knockdown at the transcript level was quantified by quantitative PCR with and without 4 h of IL1B treatment. (n = 4) (C) NFE2L2 induction by IL1B and its knockdown using siRNA at the protein level was detected by immunoblot by using antibodies against NFE2L2 or GAPDH to control for protein loading with or without 4 h of IL1B treatment. (n = 4) (D) CM- H2DCFDA positive cell count with and without 4 h of IL1B treatment in PHM1–31cells transfected with siRNA (scrambled, si1, si2 or si3). (n = 3) (E) Relative percentage of ROS+ cells in NFE2L2 siRNA (scrambled, si1, si2 and si3) compared to control (scrambled) with and without 4 h of IL1B treatment. (n = 3) Data represent mean values ± SEM; unpaired t-test; *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001.

Article Snippet: Chromatin was immunoprecipitated by incubating it overnight at 4 ◦C with 5.55 μg of NFE2L2 antibody (D1Z9C) XP® Rabbit mAb (Cell Signaling Technology, 12721) or normal rabbit IgG (Cell Signaling Technology, 2729) as negative control.

Techniques: Knockdown, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Control, Cell Counting

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: The NFE2L2 (NRF2) transcription factor controls genes involved in the oxidative stress response and inflammation in myometrial cells.

doi: 10.1016/j.bbamcr.2025.119985

Figure Lengend Snippet: Fig. 3. Control of HMOX1, FTH1 and AKR1B1 by NFE2L2 in PHM1–31 myometrial cells. PHM1–31 cells were transfected with siRNA (scrambled, si1, si2 or si3) analyzed in the presence or absence of IL1B (10 ng/ml). The regulation of HMOX1 (A), FTH1 (B) and AKR1B1 (C) mRNA expression upon NFE2L2 knockdown was quantified by quantitative PCR with and without 4 h of IL1B treatment. (n = 4). (D) The regulation of HMOX1, FTH1 and AKR1B1 protein level was detected by immunoblot by using antibodies against HMOX1, FTH1, AKR1B1 or GAPDH to control for protein loading at 0, 4 and 8 h of IL1B treatment. (n = 4). Data represent mean values ± SEM; unpaired t-test; *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001.

Article Snippet: Chromatin was immunoprecipitated by incubating it overnight at 4 ◦C with 5.55 μg of NFE2L2 antibody (D1Z9C) XP® Rabbit mAb (Cell Signaling Technology, 12721) or normal rabbit IgG (Cell Signaling Technology, 2729) as negative control.

Techniques: Control, Transfection, Expressing, Knockdown, Real-time Polymerase Chain Reaction, Western Blot

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: The NFE2L2 (NRF2) transcription factor controls genes involved in the oxidative stress response and inflammation in myometrial cells.

doi: 10.1016/j.bbamcr.2025.119985

Figure Lengend Snippet: Fig. 4. Binding of NFE2L2 transcription factor to HMOX1, FTH1 and AKR1B1 loci in PHM1–31 myometrial cells. Location of NFE2L2 binding sites at the HMOX1, FTH1 and AKR1B1 loci were established using the UCSC genome browser database Encode 3 DNA binding data for NFE2L2 in HeLa-S3 (ENCFF712LMP) [27] and HepG2 (ENCFF297BXM) cells [28]. Chosen peak is shown with rectangle on top of peak representing statistically significant peak call, the darker rectangle being more significant. ChIP analysis of NFE2L2 transcription factor binding to HMOX1 (A), FTH1 (B) and AKR1B1 (C) locus. PHM1–31 cells were treated (or not) with 10 ng/ml IL1B for 4 h and analyzed by chromatin immunoprecipitation using antibodies against NFE2L2 or Normal Rabbit IgG #2729 to control for non-specific binding. (n = 3) Data represent mean values ± SEM; unpaired t-test; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Chromatin was immunoprecipitated by incubating it overnight at 4 ◦C with 5.55 μg of NFE2L2 antibody (D1Z9C) XP® Rabbit mAb (Cell Signaling Technology, 12721) or normal rabbit IgG (Cell Signaling Technology, 2729) as negative control.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Control