Intracerebroventricular (i.c.v) drug administration was performed as previously described (He et al., 2015 (link)). Rats were placed in a stereotaxic apparatus under anesthesia with 2.5% isoflurane in in 70/30 % medical air/oxygen. The needle of a 10 μL Hamilton syringe (Microliter701; Hamilton Company, Reno, NV, USA) was inserted into the right lateral ventricle through a burr hole using the following coordinates relative to bregma: 1.5 mm posterior, 0.9 mm lateral, and 3.3 mm below the horizontal plane of the bregma. Drugs and siRNA were infused directly into the lateral ventricles at a rate of 1 μL/min by a pump. The needle was retracted 5 minutes after the injection when the burr hole was plugged with bone wax immediately. LY294002 (Selleck Chemicals, Houston, USA) was prepared at 50 mmol/L in PBS (contains 25% DMSO), and 5 μl of the DMSO and LY294002 were infused 30 minutes before SAH induction in the SAH+Ex-4+DMSO and SAH+Ex-4+LY294002 rats, respectively. GLP-1R siRNA and scrambled RNA (OriGene Technologies, Rockville, MD, USA) were prepared at 500 pmol in RNAse free suspension buffer and were infused (5 μl of the siRNAs) 48 hours before SAH modeling in the SAH+Ex-4+GLP-1R siRNA and SAH+Ex-4+Scr siRNA rats, respectively.
Ly294002
Manufactured by Selleck Chemicals
Sourced in United States, China, Germany, United Kingdom, Japan
LY294002 is a chemical compound used in research laboratories. It is a potent and selective inhibitor of the PI3 kinase enzyme.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (56 mentions)
Mk 2206, by Selleck Chemicals (39 mentions)
U0126, by Selleck Chemicals (38 mentions)
Rapamycin, by Selleck Chemicals (33 mentions)
Pd98059, by Selleck Chemicals (32 mentions)
Sb203580, by Selleck Chemicals (28 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (28 mentions)
Sp600125, by Selleck Chemicals (27 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (19 mentions)
Mg132, by Selleck Chemicals (17 mentions)
Penicillin, by Thermo Fisher Scientific (17 mentions)
Streptomycin, by Thermo Fisher Scientific (16 mentions)
Sch772984, by Selleck Chemicals (14 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (14 mentions)
β actin, by Cell Signaling Technology (13 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (13 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (13 mentions)
P akt, by Cell Signaling Technology (10 mentions)
Gefitinib, by Selleck Chemicals (10 mentions)
Cleaved caspase 3, by Cell Signaling Technology (10 mentions)
465 protocols using ly294002
1
Intracerebroventricular Drug Administration Protocol
2
Investigating PI3K and JAK2 Inhibition in Cerebral I/R
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To Figureure out the role of the PI3K pathway following cerebral I/R, rats in the Res30 + LY294002 group were pretreated with LY294002 (Selleckchem, Houston, USA), an effective inhibitor of PI3K, as previously described.20 (link) Prior to surgery, dimethyl sulfoxide (DMSO) and ethanol (ETOH) were used as solvents for LY294002, dissolved to a concentration of 20 mM. Animals were anesthetized (7% chloral hydrate, 350 mg/kg, IP) and fixed on a stereotaxic apparatus. The skull was exposed as follows: anteroposterior (AP), 0.8 mm posterior to bregma; mediolateral (ML), 1.4 mm away from midline on the right side; dorsoventral (DV), 3.6 mm deep into the skull surface. The preparation of LY294002 and the vehicle was performed by the same researcher who was responsible for the drug administration. At 30 min before surgery, intracerebroventricular injection of 5 μl LY294002 solution or vehicle (DMAO + ETOH) into the ischemic side was performed.21 (link) We examined the effects of low (2 μl, 20 nM/ml), medium (4 μl, 20 nM/ml), and high (6 μl, 20 nM/ml) dosages of AG490 (JAK2 inhibitor; Selleckchem, Houston, USA) on cerebral I/R injury to identify the optimal dosage (6 μl, 20 nM/ml) for maximizing the inhibiting effects. The Res30 + AG490 group was subjected to the same procedure as the Res30 + LY294002 group.
3
Modulating EPCs' Hypoxic Stress Response
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To simulate the ischemic and hypoxic microenvironment in CHD, EPCs were exposed to hypoxic conditions (1% O2, 95% N2, 5% CO2) and starved with 1% fetal bovine serum in EBM-2 medium for 24 h. Cells incubated under normoxic conditions (95% O2, 5% CO2) and EGM-2 MV medium for 24 h were used as controls. The EPCs were then divided into six groups: (1) control group; (2) hypoxia group; (3) hypoxia + ECSW group (hypoxia injury model and for extracorporeal cardiac shock waves treatment); (4) hypoxia + LY294002 + ECSW group (hypoxia injury model treated with LY294002 (selleck, Houston, Texas, USA; 100 μM) to block PI3K for 6 h before ECSW treatment); (5) hypoxia + MK-2206 + ECSW group (hypoxia injury model treated with MK-2206 (selleck, Houston, Texas, USA; 20 μM) to block Akt for 6 h before ECSW treatment); (6) hypoxia + L-NAME + ECSW group (hypoxia injury model treated with L-NAME (selleck, Houston, Texas, USA; 1 mM) to block eNOS for 6 h before ECSW treatment). ECSW treatment was measured using the MODULITH SLC instrument (STORZ MEDICAL, Switzerland) with an energy of 0.09 mJ/mm2 and a total of 500 shots.
4
Regulation of PTEN Protein Stability
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The human GCs line KGN was cultured in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12) (Gibco, Grand Island, NE, United States) containing 10% charcoal-stripped fetal bovine serum (Gibco) and 1% antibiotics (mixture of penicillin, streptomycin, and neomycin; Gibco) in a 37°C and 5% CO2 incubator (Thermo Fisher Scientific, Waltham, MA, United States). Cells were passaged every 2 days. For MG132 (100 μM, Selleck Chemicals, Houston, TX, United States) treatment, cells were harvested after 6 h of treatment. In addition, cells were treated with 100 μM cycloheximide (CHX, Selleck Chemicals, Houston, TX, United States) and collected every 12 h to assess PTEN protein stability. For inhibition of PI3K/AKT signaling, 30 μm of LY294002 (Selleck Chemicals), a specific inhibitor that blocks the PI3K/AKT pathway, was added to the cells for 12 h. Since MG132, CHX, and LY294002 were all dissolved in DMSO, an equal volume of DMSO was added to the control cells.
5
Cell Signaling Pathway Inhibition
6
Protective Effects of PNS against OGD/R-Induced Injury
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The present study was divided into five groups: (1) Control group, bEnd.3 cells were cultured under normal culture condition for the same period of time with OGD/R insult; (2) Model group (OGD/R group), bEnd.3 cells were insulted by oxygen-glucose deprivation/reperfusion; (3) Low PNS group, bEnd.3 cells were treated with 200 μg/mL PNS for 12 h before OGD; then treated with 200 μg/mL PNS for another 12 h in reperfusion period; (4) Middle PNS group (300 μg/mL PNS); (5) High PNS group (400 μg/mL PNS).
For PI3K/Akt inhibition, the PI3K inhibitor LY294002 (Selleckchem, Burlington, NC, USA) was added to the culture medium 2 h before PNS treatment and together with PNS before OGD/R. Experimental timelines of PNS with/without LY294002 treatment in bEnd.3 cells exposed to OGD/R in different experiments were summarized inFigure 8 .
For PI3K/Akt inhibition, the PI3K inhibitor LY294002 (Selleckchem, Burlington, NC, USA) was added to the culture medium 2 h before PNS treatment and together with PNS before OGD/R. Experimental timelines of PNS with/without LY294002 treatment in bEnd.3 cells exposed to OGD/R in different experiments were summarized in
7
Aging Nerve Cell Culture with EPO
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Cells in the control group were cultured for a total of 22 days and represented aged nerve cells. In the EPO group, starting on day 11, the culture medium was supplemented with EPO (final concentration, 100 µ/ml; PeproTech, Rocky Hill, Connecticut, USA). In the EPO + LY294002 group starting on day 11, the culture medium was supplemented with EPO (final concentration, 100 µ/ml; PeproTech) and LY294002 (final concentration, 5 μM; vehicle: DMSO, the final concentration of DMSO was 0.05%; Selleck, Houston, Texas, USA). In the LY294002 group, on day 11, the culture medium was supplemented with LY294002 (final concentration, 5 μM; Selleck).
8
Therapeutic Potential of C16 Peptide in NMO
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A total of 150 adult male Lewis rats weighing 250–300 g were obtained from the Laboratory Animal Services Centre of Zhejiang University, China. Eighteen rats were included in the untreated control group, and 132 rats were included in the NMO group.
The NMO rats were randomly divided into four groups: the vehicle-treated group (n = 33), wherein the rats were intravenously injected with 1 ml of phosphate-buffered saline (PBS) daily for 2 weeks; the C16-treated group (n = 33), wherein the rats were intravenously injected with 2 mg of C16 peptide (Shanghai Science Peptide Biological Technology Co., Ltd., Shanghai, China) daily for 2 weeks; the C16 and Tie2 kinase inhibitor-treated group (Tie2 KI + C16 group; n = 33), wherein the rats were intravenously injected with 2 mg of C16 peptide daily for 2 weeks and intraperitoneally injected with 25 mg/kg of the Tie2 kinase inhibitor (Selleck, Shanghai, China) daily for 2 weeks; and the C16 peptide and LY294002-treated group (LY294002 + C16 group; n = 33), wherein the rats were intravenously injected with 2 mg of C16 peptide daily for 2 weeks and intraperitoneally injected with 100 mg/kg of the class I PI3K inhibitor LY294002 (Selleck, Shanghai, China) daily for 2 weeks.
The NMO rats were randomly divided into four groups: the vehicle-treated group (n = 33), wherein the rats were intravenously injected with 1 ml of phosphate-buffered saline (PBS) daily for 2 weeks; the C16-treated group (n = 33), wherein the rats were intravenously injected with 2 mg of C16 peptide (Shanghai Science Peptide Biological Technology Co., Ltd., Shanghai, China) daily for 2 weeks; the C16 and Tie2 kinase inhibitor-treated group (Tie2 KI + C16 group; n = 33), wherein the rats were intravenously injected with 2 mg of C16 peptide daily for 2 weeks and intraperitoneally injected with 25 mg/kg of the Tie2 kinase inhibitor (Selleck, Shanghai, China) daily for 2 weeks; and the C16 peptide and LY294002-treated group (LY294002 + C16 group; n = 33), wherein the rats were intravenously injected with 2 mg of C16 peptide daily for 2 weeks and intraperitoneally injected with 100 mg/kg of the class I PI3K inhibitor LY294002 (Selleck, Shanghai, China) daily for 2 weeks.
9
Modulating FABP5 and PI3K/AKT in KGN cells
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KGN cells from our laboratory were used in cell culture experiments. The KGN cell line was grown in DMEM/F12 medium (Corning, New York, USA) supplemented with 8% (v/v) bovine calf serum (Sigma, Shanghai, China), 100 U/ml penicillin and 100 mg/ml streptomycin (HyClone, South Logan, UT, USA) at 37 °C with 5% CO2. Forty-eight hours after transfection with the myc-FABP5 plasmid or FABP5 siRNA, KGN cells were treated with 10 μM LY294002 (Selleck, Texas, USA) or SC79 (Selleck, Texas, USA) for another 48 h; these cells were referred to as myc-FABP5 + LY294002 or siFABP5 + SC79 cells, respectively.
10
Conditional gene knockout in mice
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Znhit1fl/fl mice were generated by Model Animal Research Center of Nanjing University (Nanjing, China) [9 (link)]. Mx1-cre mice were obtained from the Jackson Laboratory. H2afvfl/fl/H2afzfl/fl mice were obtained from RIKEN BioResource Center. All strains were maintained in C57BL/6 background.
For Cre induction, 6–8 weeks old mice were intraperitoneally injected with 300 μg polyinosinic–polycytidylic acid (pIpC) (Novus) every 2 days for three times. For LY294002 administration, mice were daily injected with 2 mg of LY294002 (Selleck) for 7 days after pIpC treatment.
All breeding and experimental procedures were performed in accordance with the relevant guidelines and regulations and with the approval of the Animal Care and Use Committee at Fudan University.
For Cre induction, 6–8 weeks old mice were intraperitoneally injected with 300 μg polyinosinic–polycytidylic acid (pIpC) (Novus) every 2 days for three times. For LY294002 administration, mice were daily injected with 2 mg of LY294002 (Selleck) for 7 days after pIpC treatment.
All breeding and experimental procedures were performed in accordance with the relevant guidelines and regulations and with the approval of the Animal Care and Use Committee at Fudan University.
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