After each treatment, the cells were washed with PBS, fixed in 4% paraformaldehyde for 30 min at room temperature, blocked with 5% (w/v) bovine serum albumin (BSA, Sigma Aldrich, USA) in PBST, and immunostained with anti-Nrf2 (1:100 dilution; Abcam, USA), anti-SIKE (1:100 dilution; Thermo Fisher Scientific, USA), anti-NF-κB (1:100 dilution; Abcam) and anti-p-TBK1 (1:100 dilution; Cell Signaling Technology, USA) antibody overnight at 4 °C, followed by incubation with a goat anti-mouse Alexa Fluor-488-conjugated secondary antibody and/or goat anti-rabbit Alexa Fluor-594-conjugated secondary antibody (Abcam). After washing with PBS, the cells were stained with DAPI (Beyotime, Shanghai, China) and observed under a fluorescence microscope. Immunofluorescence staining for liver sections was performed using anti-Nrf2 (1:100 dilution; Abcam) and anti-SIKE (1:100 dilution; Thermo Fisher Scientific) as described for cells with little modification. Immunofluorescence images were obtained using a fluorescence microscope. Images were analyzed with Image J software.
Anti nrf2
Manufactured by Abcam
Sourced in United Kingdom, United States, China, Germany, Spain
Anti-Nrf2 is a primary antibody used in laboratory research. It binds and detects the Nrf2 protein, which plays a role in the regulation of antioxidant response elements. This product can be used in various applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to study the expression and localization of Nrf2 in different cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti ho 1, by Abcam (36 mentions)
Anti nqo1, by Abcam (17 mentions)
Anti bcl 2, by Abcam (10 mentions)
Anti gapdh, by Cell Signaling Technology (9 mentions)
Anti β actin, by Cell Signaling Technology (9 mentions)
Pvdf membrane, by Merck Group (9 mentions)
Anti bax, by Abcam (8 mentions)
Anti gapdh, by Abcam (8 mentions)
Anti β actin, by Abcam (8 mentions)
Anti keap1, by Abcam (7 mentions)
Anti lamin b1, by Abcam (6 mentions)
Anti nf κb, by Abcam (6 mentions)
Anti p38, by Abcam (6 mentions)
Bovine serum albumin (bsa), by Merck Group (6 mentions)
Anti β actin, by Santa Cruz Biotechnology (5 mentions)
Anti flag, by Merck Group (5 mentions)
Anti p p38, by Cell Signaling Technology (5 mentions)
Anti β actin, by Merck Group (5 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (5 mentions)
Anti caspase 3, by Abcam (4 mentions)
161 protocols using anti nrf2
1
Immunofluorescence Analysis of Cellular Signaling
2
Extraction and Quantification of Nrf2
3
Protein Expression Analysis in Rat Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The primary antibodies, including anti-caspase-3, anti-caspase-9, anti-Nrf2, anti-NQO1, anti-HO-1, anti-NF-κB p65, and anti-actin, were purchased from Abcam (Cambridge, UK). After 24 h of reperfusion, the rat brain samples were homogenized, washed with phosphate buffered saline (PBS), and then lysed with radio immunoprecipitation assay (RIPA) lysis buffer. The total protein content was determined using a BCA kit (Solarbio, Beijing, China). After centrifugation at 12,000 rpm for 10 min, the supernatants were harvested for the measurement of protein concentration. The protein was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Solarbio, Beijing, China) and then transferred to polyvinylidene difluoride (PVDF) membranes (Solarbio, Beijing, China). The membranes were blocked with 5% nonfat dry milk and incubated overnight at 4 °C with anti-caspase-3, anti-caspase-9, anti-Nrf2, anti-NQO1, anti-HO-1, anti-NF-κB p65, and anti-actin. Following that, the membranes were washed with tris-buffered saline and Tween 20 (TBST) buffer. The secondary antibody was added to the membranes. The blots were detected with an ECL detection kit (Solarbio, Beijing, China). Actin was served as a loading control.
4
Oxidative Stress Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti caspase 3, anti HO1, anti Nrf2, anti SOD2, anti actin, anti lamin B1 and anti GCLC antibodies were purchased from Abcam (Cambridge, MA, USA). K2Cr2O7 and all other reagents were bought from Sisco Research Laboratory (Mumbai, India).
5
Investigating TMZ-Induced Stress Response in NK and CD8+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NK cell and CD8 T cell enrichment were performed using the CD56+ CD16+ NK and CD8+ T Cell Isolation Kit (Miltenyi Biotec), respectively. After magnetic cell separation, NK and CD8+ T cells were seeded in 6-plate wells cells at the density of 106 cells/well and treated with 25 μM of TMZ or vehicle (DMSO), for 10 and 30 min, were washed with cold PBS and lysed in a buffer supplemented with protease and phosphatase inhibitors. Membranes with transferred proteins were incubated with the primary antibody anti-pNRF2 (phosphoSer40, 1:5000, Abcam), anti-NRF2 (1:1000, Abcam) or anti-vinculin (1:10,000). The primary antibody incubation was followed by incubation with peroxidase conjugated to the secondary antibody (anti-rabbit, 1:10,000). A chemiluminescence reaction using the ECL Plus kit (GE Healthcare, Chicago, IL, USA) was detected using G: BOX iChemi system (Syngene, Cambridge, UK).
6
Oxidative Stress and Inflammation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DHC was purchased from Anyang General International (Henan, China) and the purity was 96.7% (from HPLC analysis). Anti-NOX2, anti-NOX4, anti-Nrf2, anti-NQO1 and anti-VR1 were purchased from Abcam (Abcam, MA, USA). Anti-MMP-9 were purchased from cell signaling (Danvers, MA, USA). Anti-NF-kB, anti-claudin, anti-occludin, and anti-β-actin were purchased from Millipore (Millipore, MA, USA). Anti-mouse IgG peroxidase conjugated secondary antibody and anti-rabbit IgG peroxidase conjugated secondary antibody were purchased from Merck Millipore (MA, USA). Commercial kits used for determining SOD and GPx activities were obtained from Cayman (Cayman Chemicals, Ann Arbor, MI, USA). All other reagents were obtained from Sigma (St. Louis, MO).
7
Immunofluorescence Staining of ApoE and Nrf2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunofluorescence staining, tissue sections were deparaffinized by xylene, rinsed, and blocked with goat serum (Boster Biological Technology, Pleasanton, California, USA) at 37°C in a humidified atmosphere for 1 h. Then sections were incubated overnight at 4°C with rabbit monoclonal anti‐ApoE (1:400; Abcam, Cambridge, UK) or rabbit polyclonal anti‐Nrf2 (1:400; Abcam). The sections were rinsed with 0.01 M PBS and incubated with goat IgG conjugated to Alexa 492 (1:200; Earthox, Shanghai, China) for 1 h at 37°C in a dark humidified chamber. Finally, nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (Beyotime Bio, Shanghai, China) for 5 min. After a final rinse, coverslips were mounted with an antifade mounting medium (Beyotime Bio), and sections were observed under a fluorescence microscope.
8
Western Blot Analysis of Hippocampal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The animals were euthanized under general anesthesia on day 3 after surgery, and total protein, nuclear protein and cytoplasmic protein were extracted from both hippocampus as described previously (Cheng et al., 2010 (link)). Western blotting procedures followed standard protocols (Shimamura et al., 2006 (link)). Equal amounts (30 μg) of total protein and nuclear protein were separated in 10% SDS-polyacrylamide gel and transferred to PVDF membranes. Primary antibodies included anti-β-actin (1:1000, Boster), anti-Histone H3 (1:1000, Millipore), anti-Nrf2 (1:1000, Abcam), anti-HO-1 (1:1000, Abcam), and anti-COX-2 antibody (1:1000, Abcam). Finally, protein bands were detected using an enhanced chemiluminescence (ECL) kit (ThermoFisher Scientific) according to the manufacturer's protocol. The IOD of each band was measured using a gel-image analyzing system (Fusion Optix, USA). β-actin was used as an internal control to normalize the target protein expression.
9
Immunohistochemical Analysis of NRF2 Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used were anti-NRF2 (Abcam Cambridge, UK), anti-phospho-histone H2A.X (Ser139), anti-phospho-ATM (Ser1981) (Merck KGaA, Darmstadt, Germany), anti phospho-ERK 1/2 (Cell Signaling Technology, Danvers, MA, USA), and anti-heme oxygenase 1 (HO-1) (Proteintech, Rosemont, IL, USA). ThermoFisher Scientific provided the Alexa Fluor-488 conjugated Goat Anti Rabbit and Alexa Fluor-555 conjugated Goat Anti Mouse secondary antibodies. The MEK 1/2 chemical inhibitor U0126 was purchased from Promega (Beijing, China). The ATM chemical inhibitor ku55933 was from Santa Cruz (Dallas, TX, USA). The chemical inducer of NRF2 tert-butylhydroquinone (tBHQ) was purchased from Sigma (Merck KGaA, Darmstadt, Germany). Hoechst 33342 and hydroethidine were purchased from ThermoFisher Scientific.
10
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nuclear proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Thermo‐Fisher). The following primary antibodies were used for western blot analysis: anti–phosphorylated c‐Jun NH2‐terminal kinase (P‐JNK; no. 9255; Cell Signaling Technology), anti‐JNK (no. 9525; Cell Signaling Technology), anti–phosphorylated extracellular signal–regulated kinase (P‐Erk) 1/2 (no. 4376; Cell Signaling Technology), anti‐Erk1/2 (no. 5013; Cell Signaling Technology), anti‐P‐p38 (no. 9216; Cell Signaling Technology), anti‐p38 (no. 9212; Cell Signaling Technology), anti‐LC3I/II (no. 4108; Cell Signaling Technology), anti‐Nrf2 (no. 137550; Abcam), anti‐p62 (no. 109012; Abcam), and anti‐Slc7a11 (no. 37185; Abcam).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!