Journal: Cell chemical biology

Article Title: Non-covalent NRF2 Activation Confers Greater Cellular Protection Than Covalent Activation

doi: 10.1016/j.chembiol.2019.07.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Goat polyclonal anti-KEAP1 , Santa Cruz Biotechnology , Cat# sc-15246, RRID:AB_2132638.

Techniques: Virus, Recombinant, Reverse Transcription, Reporter Assay, Software, Imaging, Modification

Journal: American Journal of Cancer Research

Article Title: PDCD4 inhibits lung tumorigenesis by the suppressing p62-Nrf2 signaling pathway and upregulating Keap1 expression

doi:

Figure Lengend Snippet: PDCD4 inhibits Nrf2 activity and nuclear localization. A. Western blots for Nrf2 and Keap1 in PDCD4-overexpressing cells. B. Two lung cancer cell lines (A549 and H460) stably expressing PDCD4 were co-transfected with pRL-TK Renilla and ARE (NRF2-luciferase) plasmids, and subjected to luciferase reporter activity assays. Mean ± SD shown for three independent experiments, and statistical significance was calculated by Student’s t-test (*, P<0.05; **, P<0.01). C. Subcellular localization of Nrf2 in A549 cells. A549 cells were transfected with the indicated amounts of PDCD4 WT plasmid for 24 h and then Nrf2 levels in the cytosolic and nuclear fractions of indicated cells were determined by western blotting. Lamin B was used as a nuclear protein marker, and β-actin as a loading control. D. PDCD4-overexpressing cell lines (A549) were immunostained with anti-Nrf2 antibody (green) and counterstained with DAPI (blue). E. Subcellular localization of Nrf2 in H460 cells. H460 cells were transfected with the indicated amounts of PDCD4 WT plasmid for 24 h and then Nrf2 levels in the cytosolic and nuclear fractions of indicated cells were determined by western blotting. Lamin B was used as a nuclear protein marker, and β-actin as a loading control. Scale bar; 20 µm. F. Two PDCD4-overexpressing cell lines (H460) were immunostained with anti-Nrf2 antibody (green) and counterstained with DAPI (blue).

Article Snippet: After three washes with phosphate-buffered saline, the slides were incubated for 2 h with FITC-conjugated goat anti-rabbit IgG (to detect anti-PDCD4) and RITC-conjugated goat anti-mouse IgG (to detect anti-Keap1) (Molecular Probes).

Techniques: Activity Assay, Western Blot, Stable Transfection, Expressing, Transfection, Luciferase, Plasmid Preparation, Marker

Journal: American Journal of Cancer Research

Article Title: PDCD4 inhibits lung tumorigenesis by the suppressing p62-Nrf2 signaling pathway and upregulating Keap1 expression

doi:

Figure Lengend Snippet: Interaction PDCD4 with Keap1 inhibits Nrf2-p62 signaling activation in lung cancer. A. Total protein extracts from control-A549 cells and PDCD4-overexpressing cells (A549) were immunoprecipitated with anti-PDCD4 or anti-IgG antibodies. PDCD4, Keap1, p62, and Nrf2 were detected by western blot after IP with anti-PDCD4. B. PDCD4-overexpressing A549 cells were transfected with plasmid encoding FLAG-tagged Keap1 and immunoprecipitated with anti-Keap1 antibody; western blots were performed to detect PDCD4 and Keap1 proteins. C. PDCD4-overexpressing A549 cells were immunostained with anti-PDCD4 (green), anti-Keap1 (red), and DAPI (blue). Stained cells were visualized on an Olympus confocal microscope. Scale bar; 10 µm. D. A549 and H460 cells were transiently co-transfected with plasmids encoding FLAG-tagged Keap1 and PDCD4 WT for 24 h, and then analyzed by western blotting with antibodies against Nrf2, PDCD4, Keap1, p62. E. Two lung cancer cell lines (A549 and H460) were co-transfected with FLAG-tagged Keap1 and PDCD4 WT, pRL-TK Renilla and ARE (NRF2-luciferase) plasmids, and subjected to luciferase reporter activity assays. Mean ± SD shown for three independent experiments, and statistical significance was calculated by Student’s t-test (*, P<0.05; **, P<0.01).

Article Snippet: After three washes with phosphate-buffered saline, the slides were incubated for 2 h with FITC-conjugated goat anti-rabbit IgG (to detect anti-PDCD4) and RITC-conjugated goat anti-mouse IgG (to detect anti-Keap1) (Molecular Probes).

Techniques: Activation Assay, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Staining, Microscopy, Luciferase, Activity Assay

Journal: American Journal of Cancer Research

Article Title: PDCD4 inhibits lung tumorigenesis by the suppressing p62-Nrf2 signaling pathway and upregulating Keap1 expression

doi:

Figure Lengend Snippet: Inhibition of Nrf2 signaling activates apoptosis in PDCD4/Keap1 overexpressing cells. A. HO-1 mRNA levels were analyzed by qRT-PCR in two PDCD4-overexpressing cell lines. Expression levels were normalized against GAPDH mRNA. Results were derived from three independent experiments, and are plotted as fold change relative to the corresponding values in control cells (defined as 1.0). B. HO-1 protein level was determined by western blot analysis. C. PDCD4-overexpressing A549 cells were co-transfected with FLAG-tagged Keap1 and siRNA targeting Nrf2 for 24 h, and then cell proliferation was detected using a WST-8 assay. Mean ± SD shown for three independent experiments, and statistical significance was calculated by Student’s t-test (*, P<0.05; **, P<0.01). D. PDCD4-overexpressing A549 cells were transiently co-transfected with FLAG-tagged Keap1 and siRNA targeting Nrf2 for 24 h, and then analyzed by western blotting with antibodies against cleaved-caspase-3 and cleaved-PARP. E. PDCD4-overexpressing A549 cells were transfected with FLAG-tagged Keap1 and siRNA targeting Nrf2 for 24 h, and then immunostained with anti-cleaved caspase-3 (green) and counterstained with DAPI (blue). The stained cells were visualized on an Olympus confocal microscope. Scale bar; 50 µm.

Article Snippet: After three washes with phosphate-buffered saline, the slides were incubated for 2 h with FITC-conjugated goat anti-rabbit IgG (to detect anti-PDCD4) and RITC-conjugated goat anti-mouse IgG (to detect anti-Keap1) (Molecular Probes).

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Derivative Assay, Western Blot, Transfection, Staining, Microscopy

Journal: American Journal of Cancer Research

Article Title: PDCD4 inhibits lung tumorigenesis by the suppressing p62-Nrf2 signaling pathway and upregulating Keap1 expression

doi:

Figure Lengend Snippet: Effect of PDCD4 overexpression in tumors derived from lung cancer cells. A and B. For xenograft studies, 1×106 PDCD4-overexpressing cells in a final volume of 100 µL were injected subcutaneously in the left flanks of mice. Representative tumors are shown. C. Immunohistochemical analysis of Ki-67 in tumor tissues. Dark brown color indicates the Ki-67 expression. Scale bar; 50 µm. D. Tumor weight, measured after the injection of PDCD4-overexpressing or control A549 cells into nude mice. E. Tumor tissue homogenates were subjected to western blot analysis with antibodies against cleaved-caspase-3, cleaved-PARP, Nrf2, PDCD4, Keap1 and p62. F. Tumor tissue homogenates were subjected to western blot analysis with antibodies against Vimentin, Snail, Slug and Twist1. G. Immunohistochemical analysis of PDCD4, p62 and cleaved PARP in tumor tissues. Dark brown color indicates the PDCD4, p62 and cleaved PARP expression. Scale bar; 50 µm. H. Immunohistochemical analysis of E-cadherin and Vimentin in tumor tissues. Dark brown color indicates the E-cadherin and Vimentin expression. Scale bar; 50 µm.

Article Snippet: After three washes with phosphate-buffered saline, the slides were incubated for 2 h with FITC-conjugated goat anti-rabbit IgG (to detect anti-PDCD4) and RITC-conjugated goat anti-mouse IgG (to detect anti-Keap1) (Molecular Probes).

Techniques: Over Expression, Derivative Assay, Injection, Immunohistochemical staining, Expressing, Western Blot

Journal: American Journal of Cancer Research

Article Title: PDCD4 inhibits lung tumorigenesis by the suppressing p62-Nrf2 signaling pathway and upregulating Keap1 expression

doi:

Figure Lengend Snippet: Schematic diagram of the mechanisms of PDCD4 induced cell death through suppression of p62/Nrf2 signaling in NSCLC cells. In this diagram, PDCD4 activates Keap1 by directly binding to PDCD4, leading to p62 and Nrf2 inhibition; Inhibition of p62/Nrf2 pathway promotes cell death in NSCLC cells.

Article Snippet: After three washes with phosphate-buffered saline, the slides were incubated for 2 h with FITC-conjugated goat anti-rabbit IgG (to detect anti-PDCD4) and RITC-conjugated goat anti-mouse IgG (to detect anti-Keap1) (Molecular Probes).

Techniques: Binding Assay, Inhibition

Journal:

Article Title: KEAP1 MODIFICATION AND NUCLEAR ACCUMULATION IN RESPONSE TO S-NITROSOCYSTEINE

doi: 10.1016/j.freeradbiomed.2007.10.055

Figure Lengend Snippet: HEK293H cells were transfected with HA-tagged Keap1 or with lipofectamine alone (sham) as described in Methods. (A) Treatment was with 500 µM spermine NONOate or CSNO for 10 minutes in HHBSS. Cellular lysates were immunoprecipitated with either anti-HA or anti-Keap1 and were immunoblotted with anti-HA as described in Methods. Keap1 has an apparent molecular weight of about 70 kDa. (B) Lysate was pre-incubated with Keap1 blocking peptide prior to immunoprecipitation and immunoblotting (lane1) and compared with control lysates (lane 2).

Article Snippet: Primary antibodies included rabbit polyclonal anti-Nrf2 (H-300) SCBT #sc-13032, goat polyclonal anti-Keap1 (E-20) SCBT #sc-15246, mouse monoclonal anti-HA-probe (F-7) SCBT #sc-7392, rabbit polyclonal anti-Lamin A (H-102) SCBT sc#20680 and mouse monoclonal anti-α-Tubulin (TU-02) SCBT sc#8035.

Techniques: Transfection, Immunoprecipitation, Molecular Weight, Incubation, Blocking Assay, Western Blot

Journal:

Article Title: KEAP1 MODIFICATION AND NUCLEAR ACCUMULATION IN RESPONSE TO S-NITROSOCYSTEINE

doi: 10.1016/j.freeradbiomed.2007.10.055

Figure Lengend Snippet: HEK293H cells were transfected with HA-tagged Keap1 and treated with spermine NONOate or CSNO in HHBSS. Lysates were prepared according to the MPB protocol, as described in Methods, followed by immunoprecipitation using anti-HA and immunoblotting with NeutrAvidin-HRP. (A) Time-response to 500 µM spermine NONOate or CSNO for 0–15 min. (B) Dose-response to 0–500 µM CSNO for 10 min.

Article Snippet: Primary antibodies included rabbit polyclonal anti-Nrf2 (H-300) SCBT #sc-13032, goat polyclonal anti-Keap1 (E-20) SCBT #sc-15246, mouse monoclonal anti-HA-probe (F-7) SCBT #sc-7392, rabbit polyclonal anti-Lamin A (H-102) SCBT sc#20680 and mouse monoclonal anti-α-Tubulin (TU-02) SCBT sc#8035.

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal:

Article Title: KEAP1 MODIFICATION AND NUCLEAR ACCUMULATION IN RESPONSE TO S-NITROSOCYSTEINE

doi: 10.1016/j.freeradbiomed.2007.10.055

Figure Lengend Snippet: HEK293H cells were treated with CSNO. Lysates were prepared according to the MPB protocol, followed by immunoprecipitation using anti-Keap1 and immunoblotting with NeutrAvidin-HRP or anti-Keap1. (A) Dose response to 0–500 µM CSNO for 10 min. (B) Time response to 500 µM CSNO for 0–60 min.

Article Snippet: Primary antibodies included rabbit polyclonal anti-Nrf2 (H-300) SCBT #sc-13032, goat polyclonal anti-Keap1 (E-20) SCBT #sc-15246, mouse monoclonal anti-HA-probe (F-7) SCBT #sc-7392, rabbit polyclonal anti-Lamin A (H-102) SCBT sc#20680 and mouse monoclonal anti-α-Tubulin (TU-02) SCBT sc#8035.

Techniques: Immunoprecipitation, Western Blot

Journal:

Article Title: KEAP1 MODIFICATION AND NUCLEAR ACCUMULATION IN RESPONSE TO S-NITROSOCYSTEINE

doi: 10.1016/j.freeradbiomed.2007.10.055

Figure Lengend Snippet: HEK293H cells which were transfected with HA-tagged Keap1 were treated with 500 µM CSNO for 0–120 minutes. Cytosolic and nuclear extracts were prepared, as described in Methods, and immunoblotted using anti-Keap1, anti-α-tubulin and anti-Lamin A. The cytosolic protein α-tubulin and the nuclear protein Lamin A were used as loading controls. The molecular weight of α-tubulin is 55 kDa.

Article Snippet: Primary antibodies included rabbit polyclonal anti-Nrf2 (H-300) SCBT #sc-13032, goat polyclonal anti-Keap1 (E-20) SCBT #sc-15246, mouse monoclonal anti-HA-probe (F-7) SCBT #sc-7392, rabbit polyclonal anti-Lamin A (H-102) SCBT sc#20680 and mouse monoclonal anti-α-Tubulin (TU-02) SCBT sc#8035.

Techniques: Transfection, Molecular Weight

Journal:

Article Title: KEAP1 MODIFICATION AND NUCLEAR ACCUMULATION IN RESPONSE TO S-NITROSOCYSTEINE

doi: 10.1016/j.freeradbiomed.2007.10.055

Figure Lengend Snippet: HEK293H cells which were not transfected were treated with 0 or 500 µM CSNO. Half of the cells were pre-treated with 5 ng/ml leptomycin B for 2 hours prior to the addition CSNO. After 90 minutes, lysates were prepared and immunoblotted using anti-Keap1, anti-Nrf2 and anti-Lamin A. The nuclear protein Lamin A was used as a loading control.

Article Snippet: Primary antibodies included rabbit polyclonal anti-Nrf2 (H-300) SCBT #sc-13032, goat polyclonal anti-Keap1 (E-20) SCBT #sc-15246, mouse monoclonal anti-HA-probe (F-7) SCBT #sc-7392, rabbit polyclonal anti-Lamin A (H-102) SCBT sc#20680 and mouse monoclonal anti-α-Tubulin (TU-02) SCBT sc#8035.

Techniques: Transfection

Journal:

Article Title: KEAP1 MODIFICATION AND NUCLEAR ACCUMULATION IN RESPONSE TO S-NITROSOCYSTEINE

doi: 10.1016/j.freeradbiomed.2007.10.055

Figure Lengend Snippet: Key thiols in Keap1 are modified by nitrosative stress as well as by oxidative stress and electrophiles. This alters the interaction of the Keap1-Nrf2-Cul3 complex in the cytosol and results in decreased degradation of Nrf2 and possibly of Keap1. Nrf2 accumulates in the nucleus where it forms a heterodimer with MafG and activates the ARE leading to transcriptional upregulation of cytoprotective and antioxidant genes.

Article Snippet: Primary antibodies included rabbit polyclonal anti-Nrf2 (H-300) SCBT #sc-13032, goat polyclonal anti-Keap1 (E-20) SCBT #sc-15246, mouse monoclonal anti-HA-probe (F-7) SCBT #sc-7392, rabbit polyclonal anti-Lamin A (H-102) SCBT sc#20680 and mouse monoclonal anti-α-Tubulin (TU-02) SCBT sc#8035.

Techniques: Modification

Journal: International Journal of Molecular Sciences

Article Title: Melatonin Promotes the Development of Secondary Hair Follicles in Adult Cashmere Goats by Activating the Keap1-Nrf2 Signaling Pathway and Inhibiting the Inflammatory Transcription Factors NFκB and AP-1

doi: 10.3390/ijms24043403

Figure Lengend Snippet: Schematic representation of the mode of action of melatonin on the promotion of secondary hair follicle growth and development. MT-melatonin, OS-oxidative stress, CAT -catalase, T-AOC -total antioxidant capacity, SOD -superoxide dismutase, GSHPX/GPx -glutathione peroxidase, MDA -malondialdehyde, SHF-secondary hair follicle, Keap1 -Kelch-like ECH-associated protein 1, Nrf2 -nuclear factor erythroid 2-related factor 2, NFκB -nuclear factor kappa B, SASP-senescence-associated phenotype, IL -interleukin, TIMP -tissue inhibitor of metallo proteinases, CXL/CCL -chemokine.

Article Snippet: The PVDF membrane was blocked with 5% skimmed cow’s milk, followed by rabbit anti- Nrf2 (1:2000), goat anti- Keap1 (1:2000), rabbit anti- P65 (1:2000), rabbit anti- C-fos (1:1000), rabbit anti- C-Jun (1:1000), rabbit anti- β-actin (1:6000) (Servicebio, Wuhan, China) overnight at 4 °C, then incubated with secondary antibodies.

Techniques: