Hcv ns3

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.

Crude replication complexes (CRCs) (0.1 μg/mL protein) diluted in PBS were fluorescently labeled with 0.18 mM lipophilic dye octadecyl Rhodamine (R18) for 15 minutes in a room temperature water bath with gentle sonication. 100 μL of fluorescently labeled CRC solution was incubated in a PDMS well on a piranha cleaned glass slide for 15 minutes. Unadsorbed CRCs were rinsed from the device with PBS. 100 μL of liposomes (0.2 mg/mL) containing POPC and 0.5 mol% DOPE PEG 2000 were incubated with the adsorbed CRCs to induce rupture for 20 minutes. Excess liposomes were rinsed using PBS. Bilayers were incubated with 1:3000 dilution of monoclonal anitbodies HCV NS3 (Abcam) or HCV NS5A (antibody 9E10; provided by Dr. Charles Rice, Rockefeller University) for 30 minutes. Unbound antibody was washed away with PBS and bilayers were incubated for 30 minutes with 1:3000 dilution of secondary goat-anti mouse conjugated to Oregon green (Molecular Probes).