Mog (HPLC > 98%) was obtained from Chengdu Pufei De Biotech Co., Ltd. (Chengdu, China; CAS:88901-36-4). The compound was dissolved in dimethyl sulfoxide (DMSO, 0.1% final concentration in cultural medium). DMSO, LPS (Escherichia coli Serotype 055:B5), and LY294002 (10 μM, AKT inhibitor) were purchased from Sigma Chemical Co. (St Louis, USA). 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and phosphate-buffered saline (PBS) were supplied by Coolaber (Beijing, China). BCA were obtained from Beyotime Biotechnology (Shanghai, China). ECL chemiluminescence substrates were supplied from Millipore Corporation (Billerica, USA). COX-2, iNOS, TLR4, Nrf2 HMGB1, IL-1β, IL-18, NF-κB, p-NF-κB, MyD88, p-AKT, AKT, p-AMPK, AMPK, IL-6, and TNF-α were obtained from ABclonal Technology (Wuhan, China). Rabbit polyclonal antibodies against ERK (#4695), p-ERK (#4370), JNK (#9252), p-JNK (#4668), p38 MAPK (#8690), p-p38 (#4511), IκBα (#4814), and p-IκBα (#2859) were purchased from Cell Signaling Technology (Bossdun, USA). NQO1 (ab80588), HO-1 (ab68477), GCLC (ab207777), and GCLM (ab126704) were provided by ABCAm (Cambridge, United Kingdom). Dulbecco's modified Eagle's medium (DMEM) and trypsin were provided by Gibco (USA). Fetal bovine serum (FBS) was from Biological Industries (Uruguay, South America).
Interleukin 6 (il 6)
Manufactured by ABclonal
Sourced in China, United States
IL-6 is a recombinant human interleukin-6 protein. Interleukin-6 is a cytokine that plays a key role in immune response and inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α, by ABclonal (9 mentions)
Il 1β, by ABclonal (8 mentions)
Cox 2, by ABclonal (3 mentions)
Gapdh, by ABclonal (3 mentions)
Il 10, by ABclonal (3 mentions)
Tnf α, by Proteintech (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
P akt, by ABclonal (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Nf κb, by ABclonal (2 mentions)
Arg 1, by ABclonal (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Mcp 1, by ABclonal (2 mentions)
Hmgb1, by ABclonal (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
30 protocols using interleukin 6 (il 6)
1
Mog Modulates Inflammatory Pathways
2
Protein Expression Analysis by Western Blot
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Total proteins were extracted from fresh cell lysates by RIPA buffer and examined by Western blot. Antibodies against ARRB1 (Cell Signaling Technology, 12697), GRP78 (Cell Signaling Technology, 3177), eIF2α (Cell Signaling Technology, 5324), p-eIF2α (Cell Signaling Technology, 3398), CHOP (Cell Signaling Technology, 2895), TNF-α (Abclonal, A11534), IL-1β (Abclonal, A19635), IL-6 (Abclonal, A0286), and β-actin (Sigma-Aldrich, A5441) were used as primary antibodies. ECL chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA) was used for signal visualization analysis. β-actin was used as a normalized control in the expression level analysis of each target protein.
3
Protein Expression Analysis in Lung and Gut
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The lung and gut tissues were frozen at −80°C. Proteins were extracted from lung and gut tissues using a protein extraction kit (Coolaber, China) in accordance with the manufacturer's instructions. The lung and gut tissues were homogenized on ice with RIPA buffer, and the protein concentrations were determined using a BCA protein assay kit (Coolaber, China). The protein samples were boiled in sample buffer, loaded into each lane, separated by 10% SDS-PAGE, and transferred to PVDF membranes. The membranes were washed with Tris-buffered saline with Tween 20 (TBST) three times and sealed with 5% nonfat milk for 2 h at 25°C. Subsequently, primary antibodies, including GAPDH (1:5000, ABclonal, China), MPO (1:1000, ABclonal, China), TNF-α (1:1000, ABclonal, China), IL-6 (1:1000, ABclonal, China), IL-10 (1:1000, ABclonal, China), α7nAchR (1:1000, ABclonal, China), and NF-κB p65 (1:1000, ABclonal, China) were incubated at 4°C overnight and then incubated at room temperature for 1 h. Finally, the protein levels were quantified using a chemiluminescence kit. The images were quantitatively analyzed using ImageJ analysis software.
4
Cytokine Expression Quantification in Hippocampus
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The frozen hippocampal tissue was lysed with RIPA buffer and homogenized on ice. Supernatants were collected after centrifugation at 10,000 g for 10 min and stored at freezing temperature for further analysis. The expression of cytokines was quantified using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s protocols (IL-6 Cat NO: RK00008, IL-1β Cat NO: RK00006, and TNFα Cat NO: RK00027, ABclonal Biotechnology Co., Ltd, Wuhan, Hubei Province, China). Briefly, after washing the wells of 96-well plate, 100 μL standard/sample (sample serum/hippocampus tissues) was added and incubated for 2 h at 37°C. The plate was then washed and a biotin-conjugated antibody (1:30) was added to each well. The plate was incubated for 1 h at 37°C. streptavidin-HRP was added for 30 min at 37°C. Finally, the reaction was stopped and the optical density was measured accordingly.
5
Centrifugation and Biomarker Analysis
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After the blood was placed in the test tube, centrifuge at 4000r/min for 10min at 4 °C. IL-1β (catalog no. RK00009; ABclonal, Wuhan, China), IL-6 (catalog no. RK00020; ABclonal, Wuhan, China) and C-Reactive Protein (CRP) (catalog no. RK00195; ABclonal, Wuhan, China) was purchased from ABclonal Technology Co.,Ltd. Detect its concentration according to the method provided by the kit.
6
Immunohistochemical Analysis of Myocardial Tissues
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After dewaxing and dehydration using xylene and ethanol, the myocardial tissues were repaired with sodium citrate. Following this, incubation with 3% hydrogen peroxide and bovine serum albumin (BSA) was performed to block endogenous peroxidase activity and antigen. Afterward, the cardiac tissues were coincubated with the primary antibodies overnight at 4°C. Primary antibodies, including those against TNF‐α (dilution 1:500; Proteintech), Bcl‐2 (dilution 1:500; Proteintech), IL‐6 (dilution 1:500; ABclonal), and Cleaved‐Caspase3 (dilution 1:100, CST), were employed. After rinsing with PBS, the appropriate secondary antibody was added, and the cells were cultured at 37°C for 1 h. Subsequently, the color development process utilized DAB color development solution (ab64238; Abcam), and hematoxylin was applied for counterstaining. The 3DHISTECH panoramic SCAN instrument was implemented for imaging and observation. Analysis of the findings was carried out using the National Institutes of Health's ImageJ software.
7
Colonic and Spleen Protein Analysis
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Colonic or spleen tissue lyses solution was fabricated using RIPA buffer (Signaling Technology, Inc.). The protein concentration was examined by a BCA kit (Sigma-Aldrich; Merck KGaA). Total protein (30 µg/sample) was separated via 10% SDS-PAGE and nitrocellulose membranes. We used 5% nonfat dried milk to block the membranes. The corresponding protein antibodies were as follows: ghrelin (Hua-bio, Chengdu, China; ER63531; 1/1000), GHSR (Abcam, MA, USA; ab85104; 1/1000), IL-1β (ABclonal, Wuhan, China; A20529; 1/500), IL-6 (ABclonal, Wuhan, China; A0286; 1/500), IL-10 (ABclonal, Wuhan, China; A2171; 1/500), TNF-α (ABclonal, Wuhan, China; A20851; 1/500), and β-actin (Boster, Wuhan, China; BM0627; 1/1000). Then, the membrane washing was performed with Tris-buffered saline/0. 1% Tween (TBST) and incubated for 1.5 hours with an HRP Goat anti-Rabbit IgG (Abcam, ab6721). The band visualization was carried out using the ECL system (Affinity Biosciences, Cincinnati, Ohio, USA), and as an internal control, β-actin was used.
8
Oridonin Modulates Stress Response Proteins
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WI-38 cells were first induced with 1 μM doxorubicin for 12 h, and then treated with oridonin or DMSO-containing medium for 48 h. WI-38 cells homogenized in RIPA lysis buffer (Thermal Scientific) with protease inhibitors (Roche) and 1% phosphatase inhibitors (Applygen). The protein extracts were homogenized using a Dounce homogenizer and spun at 12,000 g for 20 min at 4°C. BCA analysis (Pierce) was used for protein quantification, and then SDS-PAGE was used to separate the proteins and then transfer to a PVDF membrane (Amersham Biosciences). The botting membranes were incubated with primary antibodies against ACTIN, p-FOXO1, FOXO1, p21, p53, IL-1α or IL-1β (ProteinTech), IL-6 (ABclonal), and IL-8 (Immunoway) overnight at 4°C. Secondary goat anti-rabbit or anti-mouse IgG (Scicrest) was added for blotting. ECL Plus kit (Amersham Biosciences) was used for visualization. The optical density of each band was used to quantify relative protein level.
9
Perioperative Cerebral Oxygenation and Biomarkers
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Blood samples from the radial artery were collected immediately before induction of anesthesia (T0), 10 min (T1) and 60 min (T2) after turning over, immediately after the operation (T3), and 15 min after extubation (T4). Blood gasses were analyzed; pH, PaO2, PaCO2, and SaO2 were recorded; and PaO2/SaO2 was calculated. Simultaneously, rSO2 and the incidence of cerebral desaturation during surgery were recorded. Serum concentrations of interleukin (IL)-6, IL-10, and glial fibrillary acidic protein (GFAP) were determined by commercial IL-6 (ABclonal, Woburn, MA, USA), IL-10 (ABclonal) and GFAP (CUSABIO, Houston, TX, USA) ELISA kits, respectively.
10
Comprehensive Protein Extraction and Western Blot Analysis
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Total protein was extracted by lysing cells on ice for 30 min with RIPA buffer (Beyotime, China). Protein concentration was measured using the BCA Protein Assay Kit (Beyotime, China). Equal amounts of protein were separated by SDS-PAGE gel electrophoresis and transferred onto the polyvinylidene difluoride (PVDF) membrane. After being blocked at room temperature for 1 h, the PVDF membrane was incubated overnight with primary antibody at 4 °C. After being washed with TBST and TBS, the PVDF membrane was incubated with secondary antibody at room temperature for 1 h. Finally, the protein bands were visualized using an ECL chemiluminescence kit (Millipore, USA).
The primary antibodies were listed in the following: ACP5 (ABclonal, #A16338, 1:1000, USA), MMP9 (ABclonal, #A0289, 1:1000, USA), CTSK (ABclonal, #A5871, 1:1000, USA), CD86 (ABclonal, #A19026, 1:1000, USA), IL-6 (ABclonal, #A1570, 1:1000, USA), CD206 (ABclonal, #A11192, 1:1000, USA), ARG-1 (ABclonal, #A1847, 1:1000, USA), GAPDH (Beyotime, #AF5009, 1:1000, China).
The primary antibodies were listed in the following: ACP5 (ABclonal, #A16338, 1:1000, USA), MMP9 (ABclonal, #A0289, 1:1000, USA), CTSK (ABclonal, #A5871, 1:1000, USA), CD86 (ABclonal, #A19026, 1:1000, USA), IL-6 (ABclonal, #A1570, 1:1000, USA), CD206 (ABclonal, #A11192, 1:1000, USA), ARG-1 (ABclonal, #A1847, 1:1000, USA), GAPDH (Beyotime, #AF5009, 1:1000, China).
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