goat polyclonal anti keap1  (Santa Cruz Biotechnology)

Journal: Cancer research

Article Title: NRF2 induction supporting breast cancer cell survival is enabled by oxidative stress-induced DPP3-KEAP1 interaction

doi: 10.1158/0008-5472.CAN-16-2204

Figure Lengend Snippet: DPP3 interacts with KEAP1 in an oxidative stress-inducible manner through a highly conserved KEAP1-binding ETGE motif. (A) Composition of KEAP1 complexes isolated under non-stress and stress conditions. HeLa S3 cells stably expressing KEAP1 with FLAG-HA double tags at the C-terminus were either untreated or treated with 10 Gy ionizing radiation (IR), 100 μM tert-butylhydroxyquinone (tBHQ), 200 μM hydrogen peroxide (H2O2), or 2 mM hydroxy urea (HU). Cells were collected 2.5 hr after IR or drug treatment, and KEAP1-containing complexes were purified from the cytoplasmic and nuclear extracts of the cells by tandem affinity purification. The “Mock” purification was carried out using HeLa S3 cells without ectopic KEAP1. (B) Alignment of amino acid sequences of the “ETGE” or “ETGE”-like motifs and their immediate surrounding regions in proteins identified in the KEAP1 complexes purified under the “untreated” condition. CYT, cytosol; NUC, nucleus. (C-D) Oxidative stress-inducible interaction between DPP3 and KEAP1. Endogenous DPP3 was immunoprecipitated (IPed) from whole cell lysates of MCF7 cells treated with indicated concentrations of H2O2 for 3 hr (C) or diquat for 24 hr (D). Proteins in the IPed materials were analyzed by western blotting. (E) Kinetics of stress-induced DPP3 binding to KEAP1. MCF7 cells were treated with 200 μM H2O2 for indicated time periods, and the complex formation between DPP3 and KEAP1 was analyzed as above. (F) Requirement of the ETGE motif of DPP3 for KEAP1 binding. The wt and mutant DPP3 proteins were IPed with anti-FLAG beads from lysates of MCF7 cells stably expressing them. The IPed DPP3 and co-IPed KEAP1 were detected by western blotting.

Article Snippet: The primary antibodies used are as follows: anti-DPP3 rabbit monoclonal (ab133671, Abcam), anti-NQO1 mouse monoclonal (sc-32793, Santa Cruz), anti-NRF2 rabbit monoclonal (ab62352, Abcam), anti-KEAP1 goat polyclonal (E20, sc-15246, Santa Cruz), anti-β-Actin mouse monoclonal (sc-69879, Santa Cruz), anti-GAPDH rabbit polyclonal (sc-25778, Santa Cruz) and anti-p62 rabbit monoclonal (ab109012, Abcam).

Techniques: Binding Assay, Isolation, Stable Transfection, Expressing, Purification, Affinity Purification, Immunoprecipitation, Western Blot, Mutagenesis

Journal: Cancer research

Article Title: NRF2 induction supporting breast cancer cell survival is enabled by oxidative stress-induced DPP3-KEAP1 interaction

doi: 10.1158/0008-5472.CAN-16-2204

Figure Lengend Snippet: Overexpression of DPP3 enhances the stability of KEAP1. (A) Levels of NRF2, DPP3, KEAP1 and p62 in MCF7 cell lines overexpressing wt and mutant DPP3 proteins. β-Actin was used a loading control. (B-C) Stabilities of KEAP in the MCF7 cell lines harboring the empty vector or overexpressing wt DPP3. Cells were either untreated or treated with 50 μg/ml of cycloheximide for 2, 4 and 6 hr, and the proteins were analyzed by western blotting. The intensities of KEAP1 bands were quantified by the ImageJ software, normalized against those of GAPDH and plotted. B shows a set of representative western blots, and C shows means of the quantified results from 3 independent experiments. Error bars represent SDs. *p<0.05. (D) Effect of DPP3 depletion in on KEAP1 levels in the stable MCF7 cell lines. The cells were treated with a control siRNA or siRNAs targeting DPP3 coding sequence (CDS) or 3′-UTR, and the proteins were analyzed by western blotting.

Article Snippet: The primary antibodies used are as follows: anti-DPP3 rabbit monoclonal (ab133671, Abcam), anti-NQO1 mouse monoclonal (sc-32793, Santa Cruz), anti-NRF2 rabbit monoclonal (ab62352, Abcam), anti-KEAP1 goat polyclonal (E20, sc-15246, Santa Cruz), anti-β-Actin mouse monoclonal (sc-69879, Santa Cruz), anti-GAPDH rabbit polyclonal (sc-25778, Santa Cruz) and anti-p62 rabbit monoclonal (ab109012, Abcam).

Techniques: Over Expression, Mutagenesis, Plasmid Preparation, Western Blot, Software, Sequencing

Journal: Cancer research

Article Title: NRF2 induction supporting breast cancer cell survival is enabled by oxidative stress-induced DPP3-KEAP1 interaction

doi: 10.1158/0008-5472.CAN-16-2204

Figure Lengend Snippet: DPP3 is overexpressed human breast cancer and correlates with increased NRF2 target gene expression and poor prognosis. (A) Box-and-whisker plots indicating the median score (horizontal line), the interquartile range (IQR, box boundaries) and 1.5 times the IQR (whiskers) demonstrate significantly higher DPP3 mRNA expression in 94 human breast tumors compared to 94 matched adjacent normal tissue samples (p=1.84×10-40, paired t-test). (B-C) A Spearman rank correlation demonstrating that DPP3 mRNA expression is positively correlated with DNA copy number status in (B) 1,031 TCGA breast tumor samples (p=1.6×10-96; r=0.5871) and (C) 1,992 samples from the METABRIC cohort (p=2.8×10-77, r=0.4019). (D) A Spearman rank correlation demonstrating that DPP3 and KEAP1 expression are positively correlated (p=7.6×10-11, r=0.2009) in the TCGA cohort. DPP3 expression is positively associated with NRF2 target gene expression (p=5.4×10-14, r=0.2314) despite a negative correlation with NRF2 mRNA expression (p=2.8×10-08, r= [-0.1719]). Tumors with mutations in DPP3, KEAP1, NRF2, FH and KRAS are indicated with vertical bars. (E) Similar results as in D were observed in the METABRIC cohort (n=1,992). Breast cancer samples in D and E are ranked based on DPP3 mRNA expression; high KEAP1, NRF2 and NRF2 target gene expression is shown in red while low expression is indicated in blue. (F-H) Kaplan-Meier plots comparing disease-specific survival in human breast tumors from the METABRIC cohort based on high (top quartile) versus low (bottom quartile) DPP3 expression in (F) all tumors, (G) ER+ tumors, or (H) ER- tumors. (I-K) Kaplan-Meier plots comparing disease-specific survival in human breast tumors from the METABRIC cohort based on high (top quartile) versus low (bottom quartile) NRF2 target gene expression in (I) all tumors, (J) ER+ tumors, or (K) ER- tumors.

Article Snippet: The primary antibodies used are as follows: anti-DPP3 rabbit monoclonal (ab133671, Abcam), anti-NQO1 mouse monoclonal (sc-32793, Santa Cruz), anti-NRF2 rabbit monoclonal (ab62352, Abcam), anti-KEAP1 goat polyclonal (E20, sc-15246, Santa Cruz), anti-β-Actin mouse monoclonal (sc-69879, Santa Cruz), anti-GAPDH rabbit polyclonal (sc-25778, Santa Cruz) and anti-p62 rabbit monoclonal (ab109012, Abcam).

Techniques: Expressing, Whisker Assay

Journal: Chemico-Biological Interactions

Article Title: Semisynthetic flavonoid 7- O -galloylquercetin activates Nrf2 and induces Nrf2-dependent gene expression in RAW264.7 and Hepa1c1c7 cells

doi: 10.1016/j.cbi.2016.10.015

Figure Lengend Snippet: 7- O -Galloylquercetin ( 3 ) induces HO-1 expression and Nrf2 accumulation in RAW264.7 cells. Cells were treated for 6 h with 0.1% DMSO (control), 5 μM hemin (HM; positive control), 15 μM quercetin (QUE) or with 3.75–15 μM 3 or methyl gallate (MG). (A) After treatment, relative changes in Hmox1 mRNA levels were determined by quantitative real-time PCR with results normalized to Gapdh mRNA. Data are means ± SD of five experiments. * p < 0.05; ** p < 0.01, significantly increased versus control. (B) After treatment, protein levels of HO-1, Nrf2, Keap1 and actin in the whole cell lysates (20 μg/lane) were analyzed by Western blotting. Representative Western blots are shown.

Article Snippet: Rabbit polyclonal heme oxygenase-1 (sc-10789), rabbit polyclonal Nrf2 (sc-722), goat polyclonal Keap1 (sc-15246) and goat polyclonal actin (sc-1616) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

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

Journal: Asian-Australasian Journal of Animal Sciences

Article Title: Resveratrol-loaded Nanoparticles Induce Antioxidant Activity against Oxidative Stress

doi: 10.5713/ajas.15.0774

Figure Lengend Snippet: Activation of Nrf2-keap1 signaling pathway caused by resveratrol. Total protein lysates were prepared from A549 cells. Protein lysates (30 μg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose membranes. Nrf2 and Keap1 were detected by immunoblotting analysis. (A) A549 cells were treated with resveratrol (0, 25, and 50 μM) for 60 min. (B) A549 cells were treated with 50 μM of resveratrol for 90 min. Dimethyl sulfoxide (DMSO) were used as a solvent for resveratrol. Resveratrol (Res), Nanoparticle (Nano) and resveratrol-loaded nanoparticle (ResN) were used.

Article Snippet: The membrane was blocked with 5% skim milk in tris-buffered saline (TBS) with 0.01% Tween-20 for 1 h and incubated with rabbit polyclonal anti-Nrf2 antibody (1:250 dilution, Santa cruz, sc-13032, Dallas, TX, USA) or goat polyclonal anti-keap1 antibody (1:300 dilution Santa cruz, sc-15246, USA) for overnight.

Techniques: Activation Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Solvent