Western blots were performed as previously described (5 (link),22 (link)). Heart lysates were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The antibodies for JNK, phosphor-JNK (Thr183/Tyr185) (p-JNK), phosphor-AMPK-Thr172 (p-AMPK), AMPKα, phosphor-Acetyl-CoA Carboxylase (p-ACC), Acetyl-CoA Carboxylase (ACC), phosphor-c-Jun (p-Jun), c-Jun, phosphor- Eukaryotic elongation factor 2 (p-eEF2), phosphor- Serine/threonine-protein kinase ULK1 (Ser555), were purchased from Cell Signaling (Danvers, MA). 4 Hydroxynonenal (4 HNE) was purchased from Abcam.
4 hydroxynonenal hne
Manufactured by Abcam
Sourced in United States
4-hydroxynonenal (HNE) is a highly reactive aldehyde that is produced during the oxidation of polyunsaturated fatty acids. It serves as a marker of oxidative stress and can be used in research applications to study lipid peroxidation and related cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α, by Abcam (3 mentions)
Protein assay rapid kit, by Fujifilm (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Lamin b, by Abcam (2 mentions)
Interleukin 6 (il 6), by Abcam (2 mentions)
Anti blvra, by Santa Cruz Biotechnology (2 mentions)
Phospho enos s1177, by Santa Cruz Biotechnology (2 mentions)
Dinitrophenol dnp, by Abcam (2 mentions)
Il 1β, by Abcam (2 mentions)
3 nitrotyrosine 3 nt, by Abcam (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Ki67 sp6, by Abcam (1 mentions)
Mitochondria isolation kit for cultured cell, by Thermo Fisher Scientific (1 mentions)
Hif 1α, by Cell Biolabs (1 mentions)
Biotinylated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Diaminobenzidine dab, by Vector Laboratories (1 mentions)
Ti s inverted microscope, by Nikon (1 mentions)
Vectostain abc kit, by Vector Laboratories (1 mentions)
9 protocols using 4 hydroxynonenal hne
1
Western Blot Analysis of Signaling Pathways
2
Immunohistochemical Analysis of Tumors
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Tumours excised at 4 h were fixed in 4% paraformaldehyde for 24 h and paraffin-embedded. Consecutive sections were processed for immunohistochemical analysis of primary antibodies (1/100) as follows: Ki-67 (SP6, Abcam), hydroxynonenal (4HNE, Abcam) and cleaved-caspase-3 (Cell Signaling). Positive cells were counted in five high-power fields of three sections from each group.
3
Quantification of Cellular Oxidative Stress
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Whole-cell lysates and mitochondrial fraction were prepared, as previously described using RIPA buffer containing 0.1% SDS (Thermo Fisher Scientific, Tokyo, Japan) [51 (link)] and mitochondria isolation kit for cultured cells (Thermo Fisher Scientific), respectively. Protein assays were performed using a Protein Assay Rapid Kit (Wako Pure Chemical Corporation, Osaka, Japan). By using the extracted proteins, an ELISA kit was used to measure the concentration of 4-hydroxynonenal (HNE) (Abcam, Cambridge, MA, USA), GSH (Biomatik, Kitchener, ON, Canada), human ATG5 (Life Span Biosciences, Seattle, WA, USA), and mouse ATG5 (Bioassay Technology Laboratory, Shanghai, China) in whole-cell lysates, according to the manufacturer’s instructions.
4
Measuring HIF-1α, TFAM, and HNE
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ELISA kits were used to measure the concentration of HIF-1α (Cell Biolabs, Inc., San Diego, CA, USA), TFAM (MyBioSource, Inc., San Diego, CA, USA), and 4-hydroxynonenal (HNE) (Abcam, Cambridge, MA, USA). The assays were performed in accordance with the manufacturers’ instructions.
5
Immunohistochemical Analysis of 4-Hydroxynonenal
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After deparaffination and microwave antigen reparation, sections were pretreated with 3% H2O2 for 20 minutes to reduce the endogenous peroxidase activity. Then the sections were ordinally incubated with the blocking buffer (10% normal goat serum) at room temperature for 1 hour, then with primary antibodies of 4-hydroxynonenal (HNE) (1/100 dilution; Abcam, Cambridge, UK) at 4°C overnight, followed with biotinylated secondary antibody (1 : 200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and avidine-biotinylated peroxidase complex (Vectostain ABC-Kit, Vector Lab, Burlingame, CA, USA) at room temperature for 1 hour. Coloration of sections was processed with diaminobenzidine (DAB, Vector Laboratories, Burlingame, CA, USA) and finished with distilled water. After counterstaining with hematoxylin for 30 seconds, sections were dehydrated with graded ethanol, cleared with dimethylbenzene, and mounted with neutral gums. Figures were captured with a Ti-S inverted microscope (Nikon, Japan) and analyzed with Image-Pro Plus software (version 6.0, Media Cybernetics, USA).
6
Quantifying 4-Hydroxynonenal in Cell Lysates
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Whole cell lysates were prepared as previously described using RIPA buffer containing 0.1% SDS (Thermo Fisher Scientific) [54 (link)]. Protein assays were performed using the Protein Assay Rapid Kit (Wako Pure Chemical Corporation, Osaka, Japan). Using the extracted proteins, an ELISA kit was used to measure the concentration of 4-hydroxynonenal (HNE) (Abcam, Cambridge, MA, USA) in whole cell lysates according to the manufacturer’s instructions.
7
Western Blot Analysis of Oxidative and Inflammatory Markers
8
Comprehensive Molecular Pathway Analysis
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T10 and human wild-type αSyn were purchased from Sigma-Aldrich (St. Louis, MO, USA). 4-Hydroxynonenal (HNE) was purchased from Abcam (USA), while miR155-5p mimics, inhibitor, and negative control were purchased from Rio Bio (Guangzhou, China). The pRL-TK plasmids were purchased from GenePharma (Shanghai, China). SHIP1 siRNA and scrambled control siRNA were purchased from GeneChem (Shanghai, China). Lipofectamine 2000 and Lipofectamine RNAiMAX were purchased from Invitrogen (Carlsbad, CA, USA). TNFα and IL-1β enzyme-linked immunosorbent assay kits were purchased from Abcam (USA). Cu-CPT22, resatorvid, E6446, and 3α-aminocholestane (3AC) were purchased from MedchemExpress (New Jersey, USA). Antibodies against IRAK1, TRAF6, p-IKKα/β, IKKα/β, NF-κB (p65), p-NF-κB (phospho-p65), PI3K (p85), p-PI3K (phosphor-p85), t-Akt, p-Akt, SHIP1, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from Boster (Wuhan, China). Alexa Fluor 568 anti-rabbit secondary antibody and DAPI were purchased from Invitrogen (Carlsbad, CA, USA). Luciferase Reporter Gene Assay kit was purchased from Roche (Basel, Switzerland).
9
Western Blot Analysis of Oxidative Stress Markers
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The lysates from tissues were separated by SDS-PAGE then transferred onto nitrocellulose membranes. Membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20. Then membranes were incubated with following primary antibodies: anti-BLVRA (1:1000, Santa Cruz), eNOS (1:1000, Santa Cruz), phospho-eNOS (S1177) (1:1000, Santa Cruz), TLR4 (1:1000, Abcam), Actin (1:3000, Abcam), 3-Nitrotyrosine (3-NT, 1:1000, Abcam), 4-Hydroxynonenal (HNE, 1:1000, Abcam), dinitrophenol (DNP, 1:1000, Abcam), IL-1β (1:1000, Abcam), IL-6 (1:2000, Abcam), TNF-α (1:1000, Abcam), Lamin B (1:1000, Abcam) at 4 °C overnight followed by appropriated secondary antibodies (1: 5000, Santa Cruz) for 2 h. Immunocomplexes were visualized by ECL ECL plus Kit (GE Healthcare and Life Science). Relative density of immunoblots was analyzed by ImageJ software as described [23 (link)].
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