AG490 (Sigma, USA) was dissolved in dimethylsulfoxide (DMSO) (Sigma, USA) and diluted to final concentrations of 200 μM. Pumc-91 or pumc-91/ADM was treated with AG490 for 24 h. The AG490-treated pumc-91 (pumc-91/AG490) or pumc-91/ADM (pumc-91/ADM/AG490) were washed twice and then co-cultured with DCs for 24 h. Background control group was 1.2‰ DMSO treated with pumc-91 or pumc-91/ADM.
Ag490
Manufactured by Merck Group
Sourced in United States, Germany, China, Italy, Switzerland, Canada
AG490 is a lab equipment product manufactured by Merck Group. It functions as a tyrosine kinase inhibitor.
Automatically generated - may contain errors
Lab products found in correlation
Ly294002, by Merck Group (8 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
U0126, by Merck Group (5 mentions)
Pd98059, by Merck Group (5 mentions)
Sb203580, by Merck Group (5 mentions)
Wortmannin, by Merck Group (4 mentions)
Curcumin, by Merck Group (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Sp600125, by Merck Group (3 mentions)
Anti stat3, by Cell Signaling Technology (3 mentions)
U0126, by Cell Signaling Technology (3 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (3 mentions)
Bcl 2, by Cell Signaling Technology (3 mentions)
N acetylcysteine, by Merck Group (3 mentions)
Rapamycin, by Merck Group (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Ab76315, by Abcam (2 mentions)
Smad2 3, by Cell Signaling Technology (2 mentions)
P stat3 tyr705, by Cell Signaling Technology (2 mentions)
119 protocols using ag490
1
Modulation of Pumc-91 Cell Response via AG490 Treatment
2
Glioblastoma and Pancreatic Cancer Cell Lines
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U373, T98G, and U87 (human glioblastoma cell lines with mutant and wild type p53) and Panc1 (human pancreatic cancer cell line with mutant p53) were grown in RPMI 1640 (Thermo Fisher Scientific), 10% Fetal Bovine Serum (FBS) (Corning), L- glutamine, streptomycin (100 μg/ml) (Corning), and penicillin (100 U/ml) (Corning) in 5% CO2 at 37°C. Cells were always detached using Trypsin-EDTA solution (Biological Industries, Cromwell, CT, USA).
U373, T98G, U87, and Panc1 cells were treated with AG490 (100 μM) (Millipore) for 48 h. U373 cells were treated with lovastatin (50 μM) (Sigma Aldrich) for 24 h. U373 cells were pre-treated with bortezomib (5 nM) (Santa Cruz Biotechnology) for 30 min and then treated with AG490 (100 μM) (Millipore) for 48 h. U373 and Panc1 cells were treated with geldanamycin (100 nM) (Sigma Aldrich) for 24 h.
U373, T98G, U87, and Panc1 cells were treated with AG490 (100 μM) (Millipore) for 48 h. U373 cells were treated with lovastatin (50 μM) (Sigma Aldrich) for 24 h. U373 cells were pre-treated with bortezomib (5 nM) (Santa Cruz Biotechnology) for 30 min and then treated with AG490 (100 μM) (Millipore) for 48 h. U373 and Panc1 cells were treated with geldanamycin (100 nM) (Sigma Aldrich) for 24 h.
3
Rat Model of Subarachnoid Hemorrhage
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Rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate (0.35 ml/kg) and placed in a prone position. A rat model of SAH was induced by endovascular perforation, according to a study by Bederson et al (15 (link)). Rats in the SAH + AG490 and SAH + vehicle groups were treated with 2 ml AG490 (5 mM; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) or DMSO, respectively, via intraventricular injection 30 min prior to SAH (16 (link)). The injection site was 1.0 mm caudal to bregma, 1.5 mm lateral to midline and 4.0 mm deep from the dura. A rectal probe monitored the body temperature and a heating lamp maintained normothermia during the entire procedure.
4
Platelet Aggregation Inhibition Assay
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Platelet aggregation was detected with a turbidimetric aggregation monitoring device (AggRAM, Helena Laboratories, Texas, United States). The mice platelets were used for platelet aggregation assay. The PPP was used as a standard for 100% aggregation. PRP was pre-warmed to 37°C in a resting state and then incubated with 0.05, 0.1, 0.15, 0.2 and 0.4 μg/mL IL-9 for 2 minutes respectively. Subsequently, 3 μM adenosine diphosphate (ADP) was added to the plasma to induce aggregation. To examine the Janus kinase 2 (JAK2) inhibitor AG490 (Sigma, United States), PRP was pre-incubated with 25 µM AG490 for 0.5 hour. Untreated PRP was used as a control (CTRL). Platelet aggregability was expressed as the maximum aggregation rate within 100 seconds.
5
Anti-IL-6 Modulates Cognitive Function
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APP/PS1 or WT mice were randomized between experimental group conditions and received four unilateral i.c.v. infusions of 300 ng anti-IL-6 (R&D Systems) or an irrelevant antibody (anti-GFP, Invitrogen) every other day, in a total volume of 1.5 μl. Novel object recognition (NOR) and glucose tolerance (GTT) tests (see below) were performed 24 h after the third and fourth infusions of anti-IL-6 or anti-GFP, respectively. A total of 5 nmol of AG490 (Millipore, Burlington, MA) in a total volume of 1 μl was i.c.v. infused unilaterally into C57BL/6 mice thirty minutes after AβOs or Veh were i.c.v. infused. GTT and NOR were performed 36 and 48 h after treatments, respectively.
6
Signaling Pathway Modulation in HSFs
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HSFs were cultured in 100 mm Petri dishes. The inhibitor group was first treated with the small-molecule inhibitors SB431542, LY294002, AG490 or PD98059 for 2 h (10 μM). The inhibitors SB203583 (a p38 inhibitor), SP600125 (a JNK inhibitor), U0126 (an ERK1/2 inhibitor), AG490 (a JAK inhibitor) and BX795 (a TBK1 inhibitor) were purchased from Millipore (Billerica, MA). Then, the cells were treated with 3D-GF-PADM at 1 mg ml−1 for 24 h. After collection, total protein extraction was done with RIPA buffer containing phosphatase and protease inhibitors (Pierce, Rockford, IL, USA). After separation using a 12% SDS-PAGE denaturing gel and transfer to PVDF membranes, the membranes were blocked with 5% skim milk. The primary antibodies, including JAK2 (Abcam; ab108596), p-JAK2 (Abcam; ab32101), STAT3 (Abcam; ab68153), p-STAT3 (Abcam; ab76315) and HAS2 (Abcam; ab140671), were used at 4 °C overnight. Secondary antibody incubation was performed at room temperature for 1 h.
7
Cell Viability Assay with RSV and AG490
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Cells were cultured in Phenol Red-free RPMI-1640 medium contained dimethyl sulfoxide (DMSO, as control), RSV (Sigma, louis, USA) or AG490 (Sigma, louis, USA). Cell viability was measured by Cell Counting Kit-8 (CCK-8, DOJINDO, Japan) following manufacturer’s protocol. The Cell viability ratio was calculated as below:
Each experiment was carried out in six replicates and results were calculated over three independent experiments.
Each experiment was carried out in six replicates and results were calculated over three independent experiments.
8
Cardiac Fibroblast Stimulation Assay
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Human Cardiac Fibroblasts were obtained from Promocell and maintained in medium Fibroblasts Media 3. Cells were cultured according to the manufacturer´s instructions. Cells were used between passages 5–7. Cells were seeded into six-well plates at 90% confluence and serum starved for 12 h and then stimulated with Aldosterone (10−8 M, Sigma), Gal-3 (10−8 M, R&D Systems), CT-1 (10−7 M, R&D Systems), RCN-1 (0.01, 0.1 and 10 µg/mL, Abcam), RCN-2 (0.01, 0.1 and 10 µg/mL, Abcam), RCN-3 (0.01, 0.1 and 10 µg/mL, Abcam), Spironolactone (Spiro, 10−6M, Sigma) and Angiotensin II (Ang II, 10−9–10−7M, Sigma) for 24 hours for protein analysis. Aldosterone was prepared in ethanol at 10−2M and then diluted in culture medium to be used at 10−8M. Recombinant Gal-3 and CT-1 were reconstituted in PBS. The doses were chosen based on preliminary and previous studies6 (link),22 (link),30 .
For the intracellular pathways study, cells were treated with RCN-3 for 5, 15, 30 and 60 minutes. The following chemical inhibitors were added at 10−5 mol/L 1 hour prior to RCN-3 stimulation: Wortmannin (Sigma Aldrich), PD98059 (Sigma Aldrich) and AG-490 (Sigma Aldrich).
For the intracellular pathways study, cells were treated with RCN-3 for 5, 15, 30 and 60 minutes. The following chemical inhibitors were added at 10−5 mol/L 1 hour prior to RCN-3 stimulation: Wortmannin (Sigma Aldrich), PD98059 (Sigma Aldrich) and AG-490 (Sigma Aldrich).
9
AG490 Inhibition of STAT3 in TGF-β Signaling
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AG490 was purchased from MedChem Express Inc. Stocking solution of AG490 was prepared as 100 mmol/l using dimethylsulfoxide (DMSO) (Sigma-Aldrich; Merck KGaA) and stored at −20°C. AG490 stock solution was further diluted in cell culture medium to various concentrations (0, 12.5, 25, 50, 75 and 100 µmol/l) according to previous reports (15 (link),18 (link)-21 (link)). The final concentration of DMSO was not >0.1%. The recombinant human TGF-β was purchased from PeproTech Inc. TGF-β powder was dissolved by 10 mmol/l citric acid to prepare a liquid at 1.0 mg/ml (pH 3.0, containing 0.1% BSA). Long-term storage was achieved by refrigeration −20°C. According to the experiment requirements, TGF-β was diluted in cell culture medium to a final concentration of 5 µg/ml. TGF-β was used to treat the cells 2 h prior to the application of AG490.
Primary antibodies were purchased from Abcam, including antibodies against STAT3 (ab119352), phosphorylated (p-)STAT3 (Tyr705) (ab76315), CTGF (ab6992) and β-actin (ab179467). Horseradish peroxidase (HRP)-conjugated secondary antibodies produced in rabbit (SAB1301585) and mouse (SAB1411905) were purchased from Sigma-Aldrich; Merck KGaA. The primers used in this study were synthesized by Takara Biotechnology Co., Ltd (Dalian, China). Other chemical reagents without special indication were obtained from Sigma.
Primary antibodies were purchased from Abcam, including antibodies against STAT3 (ab119352), phosphorylated (p-)STAT3 (Tyr705) (ab76315), CTGF (ab6992) and β-actin (ab179467). Horseradish peroxidase (HRP)-conjugated secondary antibodies produced in rabbit (SAB1301585) and mouse (SAB1411905) were purchased from Sigma-Aldrich; Merck KGaA. The primers used in this study were synthesized by Takara Biotechnology Co., Ltd (Dalian, China). Other chemical reagents without special indication were obtained from Sigma.
10
Culturing HepG2 and Hep3B Cells
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HepG2 and Hep3B cell lines were obtained from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) and maintained in culture medium RPMI 1640 (Life Technologies, Rockville, MD, USA) with 10% fetal calf serum (FCS; HyClone, Logan, UT, USA). The AG490, a selective inhibitor of JAK/STAT3 activation, and luteolin were from Sigma (St. Louis, MO, USA). The anti-human IL-6 monoclonal antibody (MAB2061) was from R & D Systems, Inc. (Minneapolis, MN, USA). IL-6, INFγ, and IL-1β were from PeproTeck (Rehovot, Israel).
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