3T3-L1 preadipocytes were harvested at the indicated times and lysed in NP40 cell lysis buffer (Invitrogen, Carlsbad, CA) for co-immunoprecipitation. Immunoprecipitations were carried out using antibodies coupled to Dynabeads Protein A (Dynabeads® protein A Immunoprecipitation Kit, Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Immunocomplexes were resolved on 12% SDS-PAGE and then immunoblotted. Antibodies used for immunoprecipitation were anti-ALDH2 (Abcam, Cambridge, MA), anti-4-hydroxynonenal (Abcam, Cambridge, MA) and anti-PKCε (Santa Cruz Biotechnology, Dallas, TX).
Anti 4 hydroxynonenal
Manufactured by Abcam
Sourced in United Kingdom
Anti-4-hydroxynonenal is a primary antibody that binds to 4-hydroxynonenal (4-HNE), a reactive aldehyde generated during lipid peroxidation. It can be used as a marker of oxidative stress in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti aldh2, by Abcam (2 mentions)
Anti pkcε, by Santa Cruz Biotechnology (1 mentions)
Dynabeads protein a immunoprecipitation kit, by Thermo Fisher Scientific (1 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Np40 cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Gsk2578215a, by Bio-Techne (1 mentions)
Anti acetylated tubulin, by Merck Group (1 mentions)
Pdsred2 mito vector, by Takara Bio (1 mentions)
Lrrk2 in 1, by Bio-Techne (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Mdivi 1, by Merck Group (1 mentions)
Anti p62, by BD (1 mentions)
Immpress reagent kit, by Vector Laboratories (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Anti acsl4, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
8 protocols using anti 4 hydroxynonenal
1
Co-Immunoprecipitation of ALDH2, 4-HNE, and PKCε
2
Mitophagy and Neurodegeneration Protocol
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DMEM-F12 (Dulbecco's modified Eagle's medium), penicillin–streptomycin, gentamicin and fetal bovine serum (FBS) were purchased from Gibco-Invitrogen (Carlsbad, CA, USA). GSK 2578215A and LRRK2-IN-1 were purchased from Tocris Bioscience (Minneapolis, MN, USA). Stealth RNAi duplexes were purchased from Life Technologies (Carlsbad, CA, USA). BCA protein assay was from Pierce (Rockford, IL, USA). The pDsRed2-mito vector was provided by Clontech (BD Biosciences, San Jose, CA, USA), GFP-LC3 was provided by Dr. JM Fuentes (Universidad de Extremadura, Badajoz, Spain), Drp1-GFP was provided by T Wilson and Dr. S Strack (Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, IA, USA) and mRFP-GFP-LC3 was provided by Dr. E Kneck (Laboratory of Cellular Biology, Centro de Investigación Príncipe Felipe, Valencia, Spain). Anti-4 hydroxynonenal and LRRK2 antibodies were purchased from Abcam (Cambridge, UK); anti-p62 from BD (San Jose, CA, USA) and Alexa Fluor 488 were from Molecular Probes (Carlsbad, CA, USA), Invitrogen (Carlsbad, CA, USA); and anti-acetylated tubulin was from Sigma-Aldrich (St. Louis, MO, USA). The TUNEL method (MEBSTAIN Apoptosis Kit) was purchased from MBL (Carlsbad, CA, USA). Mdivi-1 was purchased from Sigma-Aldrich.
3
Immunodetection of 4-Hydroxynonenal in Tissues
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Specific antigens present in the tissue were unmasked by microwave pre-treatment in 0.01 M citrate buffer (pH: 6.2) 2 × 5 min at a power of 900 W. Tissue sections were incubated overnight at 4 °C with primary antibodies [polyclonal (rabbit) anti-4-Hydroxynonenal, (Abcam, Cambridge, UK)] diluted at 1:75 in PBS. After rinsing in PBS, slices were treated with the complexe anti-rabbit/peroxidase (ImmPress™ Reagent Kit; Vector, Burlingame, CA) for 30 min at room temperature. Bound peroxidase activity was visualized by precipitation of 3,3′-diaminobenzidine 0.02% in PBS containing 0.01% H2O2. Preparation was counterstained with hemalum and luxol fast blue, dehydrated and mounted with a permanent medium. The specificity of immunolabeling was ascertained on the basis of several criteria. In each case negative controls were essayed by omitting the primary or secondary antibody or by the substitution of non-immune serum for the primary antibody. No staining was observed on these sections in these conditions.
4
Western Blot Analysis of Ferroptosis Markers
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Tissues and Cells were lysed in the lysis buffer (RIPA, Beyotime, China). The protein concentrations were measured by the BCA assay kit (Beyotime, China). Equal amounts of protein were loaded in 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and were then transferred from the gel to polyvinylidene fluoride membranes (Millipore, USA). After blocking in a solution of 5% bovine serum albumin (BSA, Solarbio, China) for 1 h, the membranes were washed with TBST and then incubated with primary antibodies overnight at 4 °C. The following antibodies were used: anti-GPX4(1:2000), anti-4 Hydroxynonenal(1:1000, ab46545, Abcam, USA), anti-ACSL4(1:2000), anti-actin(1:2000, 4967, Cell Signaling Technology, USA). After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at 37 °C: goat anti-rabbit IgG antibody (1:2000, #7074, Cell Signaling Technology, USA). Bound antibodies were detected using the ECL western blotting detection system (Millipore, USA).
5
Western Blotting of Oxidative Stress Markers
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Western blotting was performed as previously described [32 (link), 33 (link)]. The following antibodies were used: anti-TNFSF11 (RANKL) (Abcam, ab9957); anti-Catalase (Cell Signaling Technology, 12,980); anti-GPX4 (Cell Signaling Technology, 52,455), anti-SOD1 (Proteintech, 67480-1-Ig); anti-4 Hydroxynonenal (Abcam, ab48506), anti-caspase 3 (Cell Signaling Technology, 9962), anti-caspase 9 (Cell Signaling Technology, 9502) and anti-α-Tubulin (Proteintech, 66031-1-Ig). Image J software was used for the quantitative analysis of each band, the relative protein expression was normalized with α-Tubulin.
6
Antibodies and Compounds for Ferroptosis Study
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The primary antibodies for SLC7A11 (12691 (Cell Signaling Technology), ab175186 (Abcam) and 26864‐1‐AP (Proteintech), Flag tag (14793; Cell Signaling Technology), ACSL4 (ab155282; Abcam), Streptavidin (3419; Cell Signaling Technology), GPX4(ab125066; Abcam), LAMP2 (66301‐1‐Ig; Proteintech), Anti‐4Hydroxynonenal (ab48506; Abcam), GAPDH (ab8245; Abcam), andβ‐actin (ab8226; Abcam) were commercially available. The following secondary antibodies were used in immunofluorescence assays: AF488‐anti‐Mouse (Invitrogen), AF594‐anti‐rabbit (Invitrogen), AF594‐anti‐mouse (Invitrogen), AF568‐anti‐rabbit (Abcam), AF647‐anti‐rabbit (Abcam), and AF647‐anti‐mouse (Abcam). Small‐molecular compounds such as Sorafenib (HY‐10201), Erastin (HY‐15763), Bafilomycin (HY‐100558), MG132 (HY‐13259), Cycloheximide (HY‐12320), Chlorquinaldol (HY‐17589A), Z‐VAD‐FMK(HY‐16658B), Necrosulfonamide (HY‐100573) and Ferrostatin‐1 (HY‐100579) were all from MCE. 2‐BP (21604) was from Sigma–Aldrich.
7
Western Blot Analysis of Protein Expression
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Proteins of both mouse tissue and HPASMCs were extracted [35 (link)] and fractionated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After that, the proteins were transferred to polyvinylidene difluoride membrane (Millipore) and blocked with 5% bovine serum albumin. Next, the membranes were incubated with the following primary antibodies: β-actin (13000; Wei'ao), rabbit anti-mouse polyclonal antibody, anti-4-hydroxynonenal (Abcam, 11000), anti-ALDH2 (Abcam, 11000), anti-cyclin D1 (CST, 11000), anti-PCNA (CST, 11000), anti-LC3 (CST, 11000), anti-p62 (CST, 11000), anti-ATG5 (Abcam, 11000), anti-ATG7 (Abcam, 11000), anti-Beclin1 (CST, 11000), anti-pPI3K (CST, 11000), anti-PI3K (CST, 11000), anti-pAKT (CST, 11000), anti-AKT (CST, 11000), anti-pmTOR (CST, 11000), anti-mTOR (CST, 11000), and anti-ERK1/2 (CST, 11000). Afterward, the membranes were washed three times using phosphate-buffered saline with Tween-20, and the samples were incubated with anti-mouse or anti-rabbit secondary antibodies (Kangcheng, 13000). Finally, the blots were analyzed using a chemiluminescence detection system (Thermo Fisher Scientific) and quantified using ImageJ gel analysis software.
8
Immunostaining of 4-HNE in Keratinocytes
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Keratinocytes were seeded on type IV collagen-precoated glass coverslips and incubated overnight. Cells were treated with various prostaglandins in keratinocyte growth medium as indicated for 24 hours. Cells were washed three times in cold PBS and fixed in 4% paraformaldehyde in PBS for 15 minutes, followed by 10 minutes incubation in 0.1% triton X-100 at room temperature. Cells were briefly rinsed in PBS and blocked with 5% normal goat serum in PBS for 30 minutes at room temperature. Cells incubated overnight at 4 C in primary antibody solution containing 1:200 anti-4 hydroxynonenal (#46545, Abcam, Cambridge, MA) in 1% bovine serum albumin in PBS. Cells were washed three times in PBS followed by 1-hour incubation with 488 Alexa Fluor-conjugated anti-rabbit IgG diluted 1:500 in 1% bovine serum albumin in PBS. Cells were then washed three times in PBS, and coverslips were mounted in DAPIcontaining mounting solution (Life Technologies, Grand Island, NY). Three representative images were acquired for each condition using Nikon Eclipse 80i fluorescence microscope (Nikon, Tokyo, Japan).
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