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2 protocols using bay x1005

1

Cerebral Ischemia Induced by tMCAO

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LTB4 (50 ng, Cayman USA) was dissolved in 0.5 μL ethanol and diluted to 1 mL using sterile PBS, and then infused at a rate of 2 mL/h (KD Scientific syringe pump) through a polyethylene catheter (PE-10) into the internal carotid artery immediately prior to tMCAO induction. Vehicle-treated controls were similarly infused with PBS containing 0.05% ethanol. BAY-X1005 (0.2 mg/kg, Tocris Biosciences USA) and LY255283 (1 mg/kg, Tocris Biosciences USA) dissolved in 1% DMSO in sterile saline were administered by intraperitoneal injection (1 mL/kg) 10 min before tMCAO.
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2

Lipoprotein Depletion and Lipid Signaling Assays

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AA-861, BWA4C, α-tocopherol, linoleic acid, arachidonic acid, dimethyl sulfoxide were from Sigma. CAY10566, Mead acid, dihomo-γ-linolenic acid, erastin, ferrostatin-1, and (1S,3R)-RSL3 were from Cayman Chemical. SC560, SC236, MK886, Zileuton, BAY-X1005, PD146176, and deferoxamine were from Tocris Bioscience. PSI-7977 (Sofosbuvir) and Glecaprevir were from Chemscene. Cell viability was determined using Cell Counting Kit-8 (DOJINDO, Japan). Lipoprotein-deprived serum (LPDS) was prepared by incubating the heat-inactivated FBS with fumed silica (Sigma, S5130) overnight, followed by removal of the silica by centrifugation at 2,000g for 20 min and filtration using a 0.22 μm filter device.
Primary antibodies to SCD (1:500 dilution, #2438) was from Cell Signaling Technology; FADS1 (1:1,000 dilution, #27533) was from Cayman Chemical; FADS2 (1:1,000 dilution, A10270) were from ABclonal; FADS2 (1:1,000 dilution, 28034–1-AP) was from Proteintech; HCV NS3 (1:500, ab13830) was from Abcam; GAPDH was from Wako (1:4,000 dilution, 016–25523). IRDye 680 or 800 secondary antibodies including #926–32211, #926–32212, #926–32214, #926–68020 and #926–68073 (1:20,000) were from LI-COR.
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