Diosmetin was purchased from Sigma-Aldrich (St. Louis, MO, USA). The enzyme-linked immunosorbent assay (ELISA) kits for interleukin (IL)-6, IL-1β, and tumor necrosis factor-α (TNF-α) were obtained from Abcam (Cambridge, UK). Akt, PI3K, and MyD88 were purchased from Cell Signaling Technology (Danvers, MA, USA) and NF-κB, nucleolin and β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Myd88
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
MyD88 is a protein that plays a central role in the innate immune response. It acts as an adapter protein, facilitating signal transduction from various Toll-like receptors and the interleukin-1 receptor. MyD88 is essential for the activation of the NF-κB and MAPK signaling pathways in response to pathogen-associated molecular patterns and inflammatory cytokines.
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Lab products found in correlation
β actin, by Cell Signaling Technology (17 mentions)
Nf κb p65, by Cell Signaling Technology (16 mentions)
Gapdh, by Cell Signaling Technology (12 mentions)
P nf κb p65, by Cell Signaling Technology (8 mentions)
P erk, by Cell Signaling Technology (8 mentions)
Traf6, by Cell Signaling Technology (7 mentions)
Nf κb, by Cell Signaling Technology (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
P iκbα, by Cell Signaling Technology (6 mentions)
P jnk, by Cell Signaling Technology (6 mentions)
P akt, by Cell Signaling Technology (5 mentions)
Nlrp3, by Cell Signaling Technology (5 mentions)
Caspase 3, by Cell Signaling Technology (5 mentions)
Tnf α, by Cell Signaling Technology (5 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
P38 mapk, by Cell Signaling Technology (4 mentions)
P erk1 2, by Cell Signaling Technology (4 mentions)
Erk1 2, by Cell Signaling Technology (4 mentions)
Interleukin 6 (il 6), by Cell Signaling Technology (4 mentions)
P pi3k, by Cell Signaling Technology (4 mentions)
104 protocols using myd88
1
Diosmetin Modulates Inflammatory Pathways
2
Whey Protein Hydrolysate Bioactivity Analysis
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WP were supplied by China National Research Institute of Food & Fermentation Industries (Beijing, China). The molecular weights of the WP were 140−1,000 Da and accounted for 92% of the total prepared WP and included a 3−6 amino acid sequence, prepared by hydrolysis of papain method. WP consist of 98.3% protein, 0.05% lipid, 4.56% ash content, and 4.21% water.
The TRIZOL Reagent Kit of superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-PX), epidermal growth factor (EGF), interferon-γ (IFN-γ), total antioxidant capacity (TAOC), Aminopeptidase N (APN), interleukin 1β (IL-1β), transforming growth factor-β (TGF-β), tumor necrosis factor-alpha(TNF-α), interleukin-6 (IL-6), zonula occluden (ZO-1), junctional adhesion molecule (JAM-A), and intercellular cell adhesion molecule-1 (ICAM-1) were purchased from Kiel biological technology Co.(Shanghai, China). Primary antibodies against TLR4, Myd88, p38MAPK, phosphor-p38MAPK, p44/42, and phosphor-p44/42 were purchased from Cell Signaling (Beverly, MA, USA). Horseradish peroxidase conjugated secondary antibodies and β-actin were purchased from Proteintech.
The TRIZOL Reagent Kit of superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-PX), epidermal growth factor (EGF), interferon-γ (IFN-γ), total antioxidant capacity (TAOC), Aminopeptidase N (APN), interleukin 1β (IL-1β), transforming growth factor-β (TGF-β), tumor necrosis factor-alpha(TNF-α), interleukin-6 (IL-6), zonula occluden (ZO-1), junctional adhesion molecule (JAM-A), and intercellular cell adhesion molecule-1 (ICAM-1) were purchased from Kiel biological technology Co.(Shanghai, China). Primary antibodies against TLR4, Myd88, p38MAPK, phosphor-p38MAPK, p44/42, and phosphor-p44/42 were purchased from Cell Signaling (Beverly, MA, USA). Horseradish peroxidase conjugated secondary antibodies and β-actin were purchased from Proteintech.
3
Immunoblotting Analysis of Key Signaling Proteins
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Western blots were performed with first antibodies listed below; p-Akt (#4060, Cell Signaling), Akt (#9272, Cell Signaling), p-Erk1/2 (#4370, Cell Signaling), Erk1/2 (#4695, Cell Signaling), p-IκBα (#9246, Cell Signaling), IκBα (sc-371, Santa Cruz), SYK (sc-1240, Santa Cruz), PLCγ1 (#5690, Cell Signaling), PIK3CD (sc-7176, Santa Cruz), RasGRP3 (#3334, Cell Signaling), PKCβI (sc-209, Santa Cruz), PKCβII (sc-210, Santa Cruz), IKKβ (sc-8014, Santa Cruz), MYD88 (#4283, Cell Signaling), NIK (#4994, Cell Signaling, protein was visualized by MG132 pretreatment.), FLAG (F3165, SIGMA), β-actin (sc-69879, Santa Cruz), p-RPS6 (#4858, Cell Signaling), RPS6 (#2317, Cell Signaling), RPL19 (sc-1000830, Santa Cruz), Ago2 (RN005M, MBL), GW182 (RN033P, MBL). Ras activation was assayed by comparing the amount of Ras-GTP and total Ras in identical cell lysates. The Ras-GTP was collected by pull-down with GST-Raf (Ras binding domain, Jena Bioscience). Alkaline phosphatase-conjugated anti-mouse and anti-rabbit secondary antibodies were from Promega. The blots were detected by BCIP/NBT substrate (Promega).
4
Antibody-based NF-κB Signaling Pathway
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Antibodies against the following proteins were purchased from Santa Cruz Biotechology, Inc (Santa Cruz, CA, USA): TLR4 (1:800), total-P65 (T-P65) (1:800), inhibitor (I)κBα (1:500), phosphorylated-P65 (P-P65) (1:300) and GAPDH (1:4000). The antibodies against myeloid differentiation factor-88 (MyD88) (1:1000), tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) (1:800) and transforming growth factor-β-activated kinase 1 (TAK1) (1:800) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
5
Protein Expression Analysis in Cultured Cells
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The cultured cells and tissues were lysed with sodium dodecyl sulfate (SDS) buffer containing protease inhibitor cocktail (Roche Life Science), and the protein concentration was detected with BCA protein assay kit (Thermo Fisher Scientific). After adjusting to the same concentration, the protein were separated by SDS‐PAGE and blotted with antibodies against Occludin, TLR4 (Abcam), E‐cadherin, NF‐κB, p‐NF‐κB, Cleaved Caspase‐1, MyD88, NLRP3, ASC (Cell Signaling Technology) and GAPDH (KangChen). Proteins were visualized using HRP‐conjugated anti‐rabbit or anti‐mouse IgG and the densities of the bands were quantified with ImageJ 1.42.
6
Forsythiaside Modulates Inflammatory Signaling
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After 2 and 4 h of incubation of Ti substrates with THP-1-deprived macrophages in the presence of different concentrations of forsythiaside, total proteins were extracted from cultured cells using radioimmunoprecipitation assay (RIPA) lysis buffer containing 0.1% phenylmethane sulfonyl fluoride (PMSF). Lysates were centrifuged at 15,000×g for 15 min, and the supernatants were collected. Protein concentration was determined using the BCA assay. Then, each protein lysate was resolved using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on 10% gels and transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% skimmed milk in Tris-buffered saline–Tween 20 (TBST) solution for 1 h and then incubated with primary antibodies IKK-α, phosphorylated IKK-α, IκB-α, phosphorylated IκB-α, MyD88, and GAPDH (Cell Signaling Technology, USA) diluted in 1% (w/v) skimmed milk powder in TBST overnight at 4 °C. Membranes were washed and incubated with the appropriate secondary antibodies. Antibody reactivity was detected by exposure in the Taton imaging system.
7
Western Blot Analysis of Liver Proteins
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Liver tissues were collected and homogenized in RIPA Lysis Buffer (Cell Signaling Technology, USA) containing PMSF and centrifuged at 12,000g at 4°C for 15 min. The concentration of protein samples was measured by BCA Protein Assay (Cell Signaling Technology, USA). A total of 35 µg protein for each sample were loaded, diluted with 5 × SDS‐PAGE Sample Loading Buffer (Beyotime, Shanghai, China), and heated at 95°C for 5 min. Then, the proteins underwent electrophoresis by 10% SDS‐PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Then, the membranes were incubated in blocking buffer for 1.5 hr. Afterward, the membranes were incubated with the primary antibodies of TLR4 (1:1,000, ProteinTech, USA), MyD88, NF‐κB p65, IκBα, and p‐IκBα (1:1,000, Cell Signaling Technology, USA), β‐actin (1:10,000, ProteinTech, USA), overnight at 4°C, washed with TBST (tris buffered saline with Tween), and incubated with secondary antibody (1:8,000, ProteinTech, USA) for 2 hr at room temperature. After five 10‐min washes, the expressions of proteins were analyzed by ECL (ProteinTech, USA) detection. The gray value of each band was measured using Image J software.
8
Antioxidant and Anti-inflammatory Mechanisms
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H2O2 (35 wt%) was purchased from Sigma−Aldrich (St. Louis, MO, USA). D−gal (≥99.0%) was obtained from Solarbio (Beijing, China). NMN (≥98.0%) and MET (≥98.0%) were obtained from Yuanye Biotechnology Co., Ltd. (Shanghai, China). According to a previously used method, SFE was isolated and purified [85 (link),86 (link)]. Primary antibodies against cleaved caspase−9, caspase−9, cleaved caspase−3, caspase−3, Cyclin D1, CDK6, Cyclin A, CDK2, Nrf2, HO−1, NQO−1, GCLM, TLR4, MyD88, p−NF−κB p65, p−IκB, and IL−6 were purchased from Cell Signaling Technology (Danvers, MA, USA). An antibody against β−actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibody HRP−conjugated goat anti−rabbit IgG and goat anti−mouse IgG were obtained from Abcam (Cambridge, MA, USA). Maintenance feed for mice (#SPF−F02−001) was purchased from SPF (Beijing) Biotechnology Co., Ltd.
9
Muscle and Metabolic Signaling Pathway Analysis
10
Western Blot Analysis of Autophagy and Signaling
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Regular Western blotting analysis was conducted as described previously [28 (link)] using the following primary antibodies: anti-light chain (LC)3 (1:1000), Beclin 1 (1:1000), TLR4 (1:1000), MyD88 (1:1000), mTOR (1:1000), phosphorylated (p)-mTOR (1:1000), p38 (1:1000), (p)-p38 (1:800), NF-kB p65 (1:1000), Akt (1:1000), p-Akt (1:2000), p-PI3K (1:1000), and PI3K (1:800) (Cell Signaling Technology, Inc., Danvers, MA, USA). Densitometry analysis was performed using the ImageJ Gel Analysis tool (NIH, Bethesda, MD, USA).
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