All chemicals, unless otherwise stated, were of the highest quality and were used as supplied. Phloretin (with ≥99% purity) was purchased from Sigma-Aldrich (USA). Purity of Phloretin was confirmed by HPLC and Mass spectrometer (KBSI). Anti- myeloid differentiation primary response 88 (MyD88), phosphorylation of transforming growth factor beta-activated kinase 1 (p-TAK1), antibodies were purchased from Abcam (UK). Anti-cyclooxygenase-2 (COX-2), hemeoxygenase-1 (HO-1), Actin, and p-NF-κB antibodies were purchased from Cell Signaling (USA).
P tak1
Manufactured by Abcam
Sourced in United Kingdom
P-TAK1 is a recombinant protein that represents the active form of the transforming growth factor-beta-activated kinase 1 (TAK1) enzyme. TAK1 is a serine/threonine protein kinase that plays a role in various cellular processes such as inflammation and stress response.
Automatically generated - may contain errors
Lab products found in correlation
P iκbα, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Phloretin, by Merck Group (1 mentions)
P nf κb, by Cell Signaling Technology (1 mentions)
P nf κb, by Santa Cruz Biotechnology (1 mentions)
Total p38, by Cell Signaling Technology (1 mentions)
Total jnk, by Cell Signaling Technology (1 mentions)
Total nf κb, by Cell Signaling Technology (1 mentions)
Total erk, by Cell Signaling Technology (1 mentions)
Ne per nuclear and cytoplasmic extraction reagents kit, by Thermo Fisher Scientific (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Radioimmunoprecipitation assay (ripa), by Merck Group (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase, by Abcam (1 mentions)
Nf κb p65, by Abcam (1 mentions)
Cleaved caspase 3, by Abcam (1 mentions)
P ikkα β, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Caspase 3, by Abcam (1 mentions)
5 protocols using p tak1
1
Phloretin Modulates Inflammatory Pathways
2
Extraction and Western Blot Analysis
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Total proteins of all cell lysates were extracted using radio immunoprecipitation buffer (RIPA, Sigma-Aldrich). The cytoplasmic and nuclear protein fractions were extracted using an NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher Scientific). Protein concentrations were measured using a BCA protein assay kit (Thermo Fisher Scientific), electroblotted to polyvinylidene fluoride membrane, and subsequently incubated with primary antibodies specific for MyD88 (Abcam, No. ab2064, 1:1,000), p-TAK1 (Abcam, No. ab109404, 1:1,000), total-ERK (Cell Signaling Technology, No. 9107, 1:1,000), p-ERK (Cell Signaling Technology, No. 9106, 1:1,000), total-JNK (Cell Signaling Technology, No. 9252, 1:1,000), p-JNK (Cell Signaling Technology, No. 4671, 1:1,000), total-p38 (Cell Signaling Technology, No. 9212, 1:1,000), p-p38 (Cell Signaling Technology, No. 9211, 1:1,000), total- NF-κB (Cell Signaling Technology, No. 9609, 1:1,000), p-NF-κB, and β-actin (Santa Cruz Biotechnology, No. sc-47778, 1:1,000). After incubation with peroxidase-conjugated secondary antibodies, protein complexes were visualized by WestGlowTM Chemiluminescent Substrate (Biomax Co., Ltd). The relative band intensities were quantified using the ImageJ software (version 1.52, NIH).
3
Western Blot Analysis of Apoptosis and Inflammatory Signaling
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After treatment, HPDLCs were collected and lysed in RIPA buffer for protein extraction, as per the product’s instructions. Following sodium dodecyl sulfate/polyacrylamide gel electrophoretic separation, the extracted proteins were transferred on to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk and then cultured with the primary antibodies against caspase-3 (Abcam), cleaved caspase-3 (Abcam), TLR4 (Abcam), p-TAK1 (Abcam), p-IKK-α/β (Abcam), p-IκBα (Abcam), NF-κB p65 (Abcam) and β-actin (Abcam) at 4°C overnight. Afterward, the membranes were immunoblotted with a secondary antibody conjugated to horseradish peroxidase (Abcam) for 1.5 h at room temperature. The immunoblots were detected by enhanced chemiluminescence from Pierce (Rockford, IL, U.S.A.) and quantified using ImageJ software (National Institutes of Health, Bethesda, MD, U.S.A.).
4
Western Blotting of Lung Signaling
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Lung homogenates were applied to a sulfate-polyacrylamide gel, and proteins were transferred to polyvinylidene difluoride membranes and incubated with primary antibodies directed against the following proteins: TAK-1, ERK1/2, p38 and JNK or the phosphorylated forms of ERK1/2, and JNK (all: Cell Signaling Technology, MA), p-TAK-1 (Abcam, United Kingdom), p-p38 (Santa Cruz, United States of America) or β-actin (Sigma Aldrich, Spain). Membranes were then incubated with secondary peroxidase-conjugated antibodies. Antibody binding was detected by an ECL system (Amersham Pharmacia Biotech, Amersham, United Kingdom), and images were acquired using Odyssey Fc System (Li-COR Biosciences, United States) and densitometry analysis performed using Quantity One software. Results were normalized by the relative expression of smooth muscle β-actin.
5
Protein Expression Analysis in HUVEC Cells
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Human umbilical vein endothelial cell was treated with the J1. After 48 hours, cell lysates were harvested, and the protein concentration was determined using a Bio‐Rad assay kit (Bio‐Rad Laboratories). Equal amounts of protein were separated by SDS‐PAGE and transferred to a 0.22‐μm polyvinylidene fluoride (PVDF) membrane (Bio‐Rad Laboratories). Then, the membrane was blocked with TBST (Tris‐buffered saline, pH 7.6, 0.05% Tween‐20) containing 5% skim milk powder for 2 hours. Membranes were then incubated overnight at 4°C with the indicated concentrations of the following primary antibodies: TRAF6, 1:1000 (Abcam); IGF‐1R, 1:1000 (Abcam); β‐actin, 1:500 (Zsgb Bio); p‐TAK1, 1:5000 (Abcam); TAK1, 1:5000 (Abcam); p‐IκBα, 1:3000 (Abcam); IκBα, 1:5000 (Abcam); p‐C‐jun, 1:5000 (Abcam); and C‐jun, 1:5000 (Abcam). After washing three times with TBST, the blot was incubated with a suitable secondary antibody conjugated to horseradish peroxidase at room temperature for 1 hour and then developed in an electrochemiluminescence Western blotting detection reagent (Beyotime). The expression level of the protein was compared to the expression level of the control based on the relative intensity of the bands.
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