Western blot analysis of whole cell extracts was performed as previously reported (18 (link)). The following primary antibodies included LKB1 (D60C5) (1:1000, #3047, Cell Signaling Technology), KEAP1, (1:1000, #10503-2-AP, Proteintech), NQO1 (A180) (1:1000, #3187 Cell Signaling Technology), NRF2 (EP1808Y) (1:1000, ab62352, Abcam), GCLC (1:1000, ab41463, Abcam) and β-actin (1:5000, Abcam) were used.
Ab41463
Manufactured by Abcam
Sourced in United States
Ab41463 is an antibody product offered by Abcam. It is a polyclonal antibody raised in Rabbit. The product is supplied as a liquid.
Automatically generated - may contain errors
Lab products found in correlation
Ab68477, by Abcam (2 mentions)
Peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Sc 2357, by Santa Cruz Biotechnology (2 mentions)
A3854, by Merck Group (2 mentions)
Ab34173, by Abcam (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Ab62352, by Abcam (1 mentions)
Keap1, by Proteintech (1 mentions)
β actin, by Abcam (1 mentions)
Immobilion p membrane, by Merck Group (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Ab37185, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
A 11122, by Proteintech (1 mentions)
Nupage 4 12 bis tris mini gel, by Thermo Fisher Scientific (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Ab15580, by Santa Cruz Biotechnology (1 mentions)
Deadend colorimetric tunel system, by Promega (1 mentions)
9 protocols using ab41463
1
Protein Expression Analysis by Western Blot
2
Analyzing Cytoprotective Protein Expression
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AREc32 cells were seeded in 6 multi-well plates (1 × 106 cells/well) for 24 h and treated with compounds 7a and 7b (30 µM) and culture medium (basal) for 24 h. Thereafter, cells were collected and lysed in ice-cold lysis buffer (10% glycerol, 137 mM NaCl, 1% Nonidet P-40, 20 mM Tris HCl pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium pyrophosphate, 1 μg/mL leupeptin and 1 mM Na3VO4). Then proteins (30 μg) were resolved by gel electrophoresis on sodium dodecyl sulfate–polyacrylamide (10% and 12%) and transferred to Immobilion-P membranes (MilliporeSigma, Madrid, Spain). The membranes were incubated with anti-GCLc (1:1000, Ab41463 (Abcam, Cambridge, MA, USA), anti-HO-1 (1:1000, ab68477, Abcam) or anti-Actin (1:100,000, A3854, Merck, Madrid, Spain). Peroxidase-conjugated secondary antibodies (1:10,000 and antirabbit: SC-2357, Santa Cruz Biotechnology, Dallas, TX, USA) were employed to detect the proteins by enhanced chemiluminescence. Band intensities corresponding to immunoblot detection of protein samples were quantitated with Fiji software [27 (link)].
3
Western Blot Analysis of Antioxidant Proteins
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AREc32 cells were collected from plates after 24 h of treatment with the selected compound at 30 µM. Then, they were lysed in ice-cold lysis buffer (1% Nonidet P-40, 10% glycerol, 137 mM NaCl, 20 mM Tris HCl pH 7.5, 1 μg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium pyrophosphate, and 1 mM Na3VO4). After this treatment, proteins (30 μg) were resolved by gel electrophoresis on sodium dodecyl sulfate–polyacrylamide (12%) and transferred to Immobilon-P membranes (MilliporeSigma, Burlington, MA, USA). The membranes were incubated with anti-GCLc (1:1000, Ab41463 (Abcam, Cambridge, MA, USA), anti−HMOX1 (1:1000, ab68477, Abcam), anti-NQO1 (1:1000, 11451-1-AP, Proteintech, Manchester, UK), or anti-β-Actin (1:100,000, A3854, Merck, Madrid, Spain). Peroxidase-conjugated secondary antibodies (1:10,000 and anti-rabbit: SC-2357, Santa Cruz Biotechnology, Dallas, TX, USA) were employed to detect the proteins by enhanced chemoluminescence. Band intensities corresponding to immunoblot detection of protein samples were quantitated with Fiji software [19 (link)].
4
Protein Extraction and Immunoblotting Protocol
5
Quantitative Analysis of Glioma Biomarkers
6
Radiation-Induced Protein Expression Analysis
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Western blotting was performed as previously described [22 (link)]. Cell extracts were collected 24 h after irradiation. Primary antibodies against GPx1 (#3206, Cell signaling), glutamate-cysteine ligase catalytic (GCLC) (ab41463, Abcam), and β-tubulin (10068-1-AP, Proteintech) and secondary anti-rabbit antibodies conjugated to horseradish peroxidase (GE Healthcare) were used. Protein bands were visualized using Chemi-Lumi One L Western blotting substrate (Nacalai Tesque), and band intensities were measured using Image Lab software (Bio-Rad). Protein expression levels were normalized to β-tubulin and are expressed relative to the control value of non-irradiated cells.
7
Protein Expression Analysis by Western Blot
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Total proteins were prepared using lysis buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, and protease inhibitor cocktail (Sigma-Aldrich). Proteins (25 µg) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes as described. After blocking, the membrane was probed with TH (1:1000; Millipore #612300), BCL2 apoptosis regulator (BCL2) (1:500; BioVision #3033, Milpitas, CA, USA), BCL2 associated X, apoptosis regulator (BAX) (1:500; BioVision #3032), microtubule associated protein 1 light chain 3 alpha (LC3) (1:3000; MBL International #PM036, Ottawa, IL, USA), nuclear factor, erythroid 2 like 2 (NRF2) (1:200; Santa Cruz Biotechnology #sc-365949), NAD(P)H quinone dehydrogenase 1 (NQO1) (1:1000; Sigma-Aldrich #N5288), glutamate-cysteine ligase catalytic subunit (GCLC) (1:1000; Abcam #ab41463, Cambridge, MA, USA), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000; MDBio #30000002, Taipei, Taiwan) at 4 °C for overnight. Then the immune complexes were detected by horseradish peroxidase conjugated goat anti-mouse (#GTX213111-01) or goat anti-rabbit (#GTX213110-01) IgG antibody (1:5000, GeneTex) and chemiluminescent substrate as described.
8
Pancreatic Protein Extraction and Western Blot
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Pancreatic tissues were frozen at -80° C until homogenization in extraction buffer (100 mg/ml) on ice. The extraction buffer contained 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Igepal® CA-630, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 50 μM sodium orthovanadate (all from Sigma-Aldrich), and a protease inhibitors cocktail (Sigma-Aldrich) at a concentration of 4 μl/ml.
The following antibodies were used: anti-PGC-1± (1/500) (2178, Cell Signaling Technology, Danvers, MA, USA), anti-GCLc (1/1000) (ab41463, Abcam, Cambridge, UK), anti-NF-º B p65 (1/1000) (8242, Cell Signaling Technology), anti-phospho-NF-º B p65 (Ser 536) (1/1000) (3039, Cell Signaling Technology), anti-histone 3 (1/1000) (ab5103, Abcam), anti-phospho-STAT3 (Tyr705) (1/1000) (9145, Cell Signaling Technology), anti-STAT3
(1/1000) (4904, Cell Signaling Technology) and anti-beta tubulin (1/1000) (ab7792, Abcam).
The following antibodies were used: anti-PGC-1± (1/500) (2178, Cell Signaling Technology, Danvers, MA, USA), anti-GCLc (1/1000) (ab41463, Abcam, Cambridge, UK), anti-NF-º B p65 (1/1000) (8242, Cell Signaling Technology), anti-phospho-NF-º B p65 (Ser 536) (1/1000) (3039, Cell Signaling Technology), anti-histone 3 (1/1000) (ab5103, Abcam), anti-phospho-STAT3 (Tyr705) (1/1000) (9145, Cell Signaling Technology), anti-STAT3
(1/1000) (4904, Cell Signaling Technology) and anti-beta tubulin (1/1000) (ab7792, Abcam).
9
Quantifying Renal GCLC Expression
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The expression of catalytic subunit of glutamate-cysteine ligase (GCLC) in remnant kidneys was analyzed by Western blotting. For this, tissue extracts were prepared in RIPA buffer containing a cocktail of protease inhibitors (Roche, Mannheim, Germany). After electrophoretic separation in a 10% acrylamide/bis-acrylamide gel, proteins were blotted on a nitrocellulose membrane (BioRad, Hercules, CA). The membrane was incubated with 5% skim milk, then with anti-GCLC (Abcam ab41463, Cambridge, MA) 1:500 or with anti-b-actin (Sigma A5441, Saint Louis, MO) 1:4000, followed by antirabbit IgG coupled to HRP (Sigma A0545)
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