Tissue samples and cells were lysed with pre-cold RIPA lysis solution (Beijing Applygen Technologies) in a glass homogenizer, and centrifuged at 12,000 g for 10 min. Protein concentration was measured using a BCA kit (Thermo Fisher Scientific). Proteins (35 μg/sample) were separated using polyacrylamide gel electrophoresis with 10% sodium dodecyl sulfate and transferred to polyvinylidene difluoride membranes (Millipore, Darmstadt, Germany). The membranes were blocked with 5% nonfat milk prior to overnight incubation at 4 °C with a primary antibody against VDBP (1:5000 dilution, cat. no. ab81307, Abcam), c-Jun N-terminal kinase (JNK) (1:1000 dilution, cat. no. #9252, Cell Signaling Technology), or phosphorylated JNK (p-JNK) (1:2000 dilution, cat. no. #9255, Cell Signaling Technology). The membranes were then incubated with an appropriate horseradish peroxidase (HRP)-labeled secondary antibody (Zhongshan Jinqiao Biotechnology) for 1 h. Protein bands of interest were visualized using an ECL kit (Thermo Fisher Scientific).
C jun n terminal kinase jnk
Manufactured by Abcam
Sourced in United Kingdom
C-Jun N-terminal kinase (JNK) is an enzyme that plays a role in cellular signal transduction pathways. JNK is part of the mitogen-activated protein kinase (MAPK) family and is responsible for the phosphorylation of the c-Jun transcription factor.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
P jnk, by Abcam (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Phosphorylated jnk p jnk, by Cell Signaling Technology (1 mentions)
Ripa lysis solution, by Applygen (1 mentions)
Horseradish peroxidase hrp labeled secondary antibody, by Zhongshan Jinqiao Biotechnology (1 mentions)
Luciferase reporter vector, by Promega (1 mentions)
Luciferase activity assay kit, by Promega (1 mentions)
Matrigel, by Bio-Rad (1 mentions)
Cox 2, by Abcam (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Transwell chamber, by Corning (1 mentions)
Horseradish peroxidase conjugated anti rabbit igg, by Merck Group (1 mentions)
β actin ab8226, by Abcam (1 mentions)
Hydroxyl free radical assay kit, by Nanjing Jiancheng (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
5 protocols using c jun n terminal kinase jnk
1
Western Blot Analysis of VDBP, JNK, and p-JNK
2
Glioma Cell Lines and Astrocyte Culture
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Human glioma cell lines U-87, C6, U-373, and M059J and human astrocytes were purchased from the American Type Culture Collection (Manassas, VA, USA). Cell culture conditions were Roswell Park Memorial Institute (RPMI) or Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), at 37°C, 5% CO2. FBS, RPMI, and DMEM were purchased from Gibco (Rockville, MD, USA). COX-2, extracellular signal-regulated kinase1 (ERK1), phosphorylated (p)-ERK1, c-Jun N-terminal kinase (JNK), and p-JNK were purchased from Abcam (Cambridge, UK). The Transwell chamber was purchased from Corning (Corning, NY, USA). Matrigel was purchased from Bio-Rad (Hercules, CA, USA). Nude mice [6 weeks old, specific pathogen free (SPF)] were provided by the experimental animal center of Sichuan University. TRIzol was purchased from Invitrogen (Carlsbad, CA, USA). Reverse transcription kits (FSQ-101) were purchased from TOYOBO Company (Kyoto, Japan). Lipofectamine 2000 and miR-26b mimic were purchased from Ji Kai Gene Company (Shanghai, P.R. China). Luciferase activity assay kit and luciferase reporter vectors were purchased from Promega (Madison, WI, USA).
3
Investigating Anti-Inflammatory Mechanisms
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All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. ISO was obtained from Baxter (Baxter Healthcare Corporation, Deerfield, IL, USA). Rabbit anti-mouse COX2, IκB kinase-β (IKKβ), NF-κB, p38 MAPK, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases 1 and 2 (ERK 1/2), β-actin, and lamin B antibodies were purchased from Abcam (Cambridge, UK). Rabbit anti-mouse phospho-IKKβ (pIKKβ, Ser180), phospho-NF-κB (Ser536), phospho-p38 MAPK (Thr180/Tyr182), phospho-JNK (Thr183/Tyr185), and phospho-ERK1/2 (Thr185/Tyr187) were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Horseradish peroxidase-conjugated anti-rabbit IgG was obtained from Chemicon (Temecula, CA, USA). ROS scavenger (Edaravone, MCI-186) was from Tocris Bioscience (Ellisville, MO, USA). p38 MAPK activation inhibitor (SB202190) and NF-κB activation inhibitor (NAI) were purchased from Biomol (Plymouth Meeting, PA, USA) and Calbiochem (Darmstadt, Germany), respectively. ZY from S. cerevisiae was dissolved in isotonic sodium chloride solution to a final concentration of 25 mg/mL. The solution was homogenized by magnetic stirring and sterilized at 100°C for 80 min. All suspensions were freshly prepared prior to use.
4
Ginsenoside Extraction and Evaluation
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5-year-old cultivated fresh ginsengs were obtained from Jingyu County, Jilin Province, China. The ginsenosides PPD, Rb1, Rf, F2, Rg3, Rh1, Ro, Rc, Rb2, Rg1, Re, Rf, Rh2 and oleanolic acid (OA) were ordered from Nanjing Jingzhu Biotechnology Corporation, China. Acetonitrile, methanol and formic acid (HPLC ≥98%) were purchased from Merck (Germany). CTX was bought from Shanghai Yuanye Bio-Technology Corporation (Shanghai, China). Commercial kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST), hydroxyl free radical assay kit, glutathione peroxidase (GSH-Px), glutathione (GSH), malondialdehyde (MDA), and total superoxide dismutase (T-SOD) were provided from Nanjing Jian Cheng Bioengineering Inc. (Nanjing, China). Radioimmunoprecipitation assay (RIPA, 89900) and phenylmethanesulfonyl fluoride (PMSF, 36978) buffer were bought from Thermo Fisher Scientific (MA, USA). The primary antibodies of c-Jun N-terminal kinase (JNK), p-JNK, extracellular signal-regulated protein kinase (ERK), p-ERK, p38, p-p38, toll-like receptor 4 (TLR4), heme oxygenase 1 (HO-1), Nrf2, the secondary antibodies of HRP-labeled goat Anti-Rabbit IgG H&L (ab205718, 1:5000) and HRP-labeled goat Anti-Mouse IgG H&L (ab6789, 1:5000) and β-actin (ab8226, 1:2000) antibodies were ordered from Abcam (Cambridge, UK). Other reagents (analytical grade) were ordered from standard commercial suppliers.
5
Palbociclib Modulates Mast Cell Signaling
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Palbociclib was purchased from Med Chem Express (Monmouth Junction, NJ). Monoclonal DNP-specific IgE, DNP-HSA, and 4-nitrophenyl N-acetyl-β-D-glucosaminide were obtained from Sigma-Aldrich (St. Louis, MO). Evans blue, formamide, toluidine blue and mast cell stabilizer ketotifen were obtained from Dalian Meilun Biotechnology Co. Ltd. (Dalian, China). Antibodies targeting the tyrosine-protein kinase Lyn, Tyr397 phosphosphorylated (p)-Lyn, mitogen-activated protein kinase (MAPK) p38, c-Jun N-terminal kinase (JNK) (Abcam, Cambridge, MA), extracellular signal-regulated kinase (ERK)1/2, p-p38 (Thr180/Tyr182), p-JNK (Thr183/Tyr185), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology, Santa Cruz, CA), and p-ERK1/2 (Thr202/Tyr204) (p-ERK1/2) (Cell Signaling Technology, Beverly, MA) were used. Fluorescein isothiocyanate (FITC)-phalloidin was purchased from Yeasen Biotech Co. Ltd. (Shanghai, China). Lyn inhibitor Bafetinib, ERK inhibitor U0126, JNK inhibitor SP600125 and p38 inhibitor SB203580 were purchased from MedChem Express (Monmouth Junction, NJ, USA). Silencing RNAs (SiRNAs) for knocking down the expression of Lyn were designed and obtained from Gene Pharma (Shanghai, China).
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