Ab2740

Sulfo-N-succinimidyl oleate (SSO, sc-208408), bafilomycin A1 (sc-201550) and siRNAs for CD36 (sc-29995), SR-BI (sc-44752 and sc-44753) and negative control (siRNA-A, sc-37007) were from Santa Cruz Biotechnology, Inc. The compounds W-9 and AP5055 were synthesized in the Medicinal Chemistry Laboratory of Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, in a purity greater than 98.5%. The structures of the compounds were confirmed with proton nuclear magnetic resonance spectroscopy and mass spectrometry spectra. Telaprevir/VX-950 (HY-10235) was from MedChemExpress, Inc. The antibody to SR-BI (NB400-104) was from Novus Biological, Inc. The mAbs to human CD36 (ab17044, ab23680, ab76521 and ab133625), HCV core (ab2740), HCV NS3 (ab13830), control IgG₁ (ab18447) and Nrf2 antibody (ab31163) were from Abcam, Co. Ltd. The mAb to beta-Actin (TA-09) was from Beijing ZSJQ-BIO, Co. Ltd. The antibodies to HCV E1 (GTX103352) and E2 (GTX103353) were from Gene Tex, Inc. The mAb to HA tag (6E2) (2367S) and the secondary antibodies (7074S and 7076S) were from Cell Signaling Technology, Inc.

All DNA and RNA oligonucleotides were purchased from TSINGKE, Co. (Wuhan, China). Anti-NCL antibody (ab129200, Abcam, UK), anti-HCV core 1b antibody (ab2740, Abcam, UK), monoclonal mouse β-actin antibody (T0022, Affinity Bioscience, USA), monoclonal mouse GAPDH antibody (60,004-1-Ig, Proteintech, USA), goat anti-rabbit immunoglobulin G (IgG, H+L) horseradish peroxidase (HRP, S0001, Affinity Bioscience, USA), and goat anti-mouse IgG (H+L) HRP (S0002, Affinity Bioscience, USA) were used in this study.

Monoclonal antibodies recognizing HCV core (ab2740) and NS3 (ab65407) were purchased from Abcam (Cambridge, UK); ApoJ (ARG62961) for IFA from Arigo Biolaboratories (Taipei, Taiwan); actin (MAB1501) from Millipore (Billerica, MA); and DsRed (tcba13674) from Taiclone Biotech Corp. (Taipei, Taiwan). Polyclonal antibodies recognizing human ApoJ for western blot (WB) analysis (ab69644) was purchased from Abcam; human ApoJ (sc-6419) for immunoprecipitation from Santa Cruz Biotechnology (Santa Cruz, CA); mouse ApoJ for WB analysis (PA5-46931) from Thermo Fisher Scientific Inc. (Waltham, MA); SOAT1 (ARG56476) and SOAT2 (ARG57814) for WB analysis from Arigo Biolaboratories; and SOAT 1 (bs-7544R) and SOAT 2 (bs-5020R) for IFA from Bioss Antibodies (Beijing, China). Goat anti-mouse Alexa-488-, and anti-rabbit Alexa-568-conjugated secondary antibodies were purchased from Thermo Fisher Scientific Inc.; Goat anti-mouse HRP- and anti-rabbit HRP-conjugated secondary antibodies were purchased from Chamot Biotechnology Co. Ltd (Shanghai, China).

Cells were differently harvested in accordance with each experiment. The J6/JFH-1-huh 7.5 cells, naïve huh 7.5 cells, or Natural killer (NK-92) cells were individually lysed by protein extraction buffer (Intron, Gyeonggi-do, Korea). Proteins in cell lysates were measured by the Bradford assay, separated by electrophoresis, and transferred to nitrocellulose membranes, which were then incubated with 1st and 2nd antibodies. Anti-HCV core Antibody 1b (#ab2740, Abcam) and anti-HCV NS3 Antibody (#ab13830, Abcam), anti-p38 (#8690, Cell Signaling, Danvers, MA, USA), anti-p-p38 (#9215, Cell Signaling), anti-Erk (#9102, Cell Signaling), anti-p-Erk (#4370, Cell Signaling) on to huh7.5 cells or J6/JFH-1-huh 7.5 cells and anti-STAT1 (#14994S, Cell Signaling) and anti-STAT5 (#9363T, Cell Signaling) onto NK-92 cells were used for first antibodies. Blots were visualized by enhanced chemiluminescent (ECL) detection solutions (Intron).

Anti septin 9 Cat#ab38314 (WB:1/500, IF:1/25), anti HCV core Cat#ab2740 (WB:1/1,000, IF:1/100), anti αtubulin Cat#ab15246 (WB:1/1,000, IF:1/100), anti septin 2 Cat#ab88657(WB:1/500, IF:1/50), mouse and rabbit anti-V5 tag Cat#ab27671 (WB:1/1,000, IF:1/400), Cat#ab9116 (WB:1/1,000, IF:1/400) respectively and anti ADFP/PLIN2 Cat#ab52355 (WB:1/2,000, IF:1/100) were from abcam; anti βtubulin Cat# T4026 (WB:1/1,000, IF:1/100) and anti TIP47/PLIN3 Cat#HPA006427 (WB:1/1,000, IF:1/100) from Sigma-Aldrich; anti-Actin Cat#sc-1616 (WB:1/1,000) and anti-PLIN2 Cat#sc-32450 (WB:1/250, IF:1/25), anti DGAT1 Cat#sc-32861 (IF:1/100) from Santa Cruz Biotechnology, anti HCV NSA Cat#HCM-131-5 (WB:1/1,000, IF:1/100) from amsbio. The mouse antibody to HCV envelope protein E2 (WB:1/500) was a gift from Dr Jean Dubuisson (CIIL, Lille, France)66 (link).

Huh7.5(NIrD) cells (2 × 10⁵ per well) were grown in 6-well plate, with a coverslip in each well. Cells were infected by HCVcc48 (link) with low MOI in the presence of Dox (2 μg/ml) for 72 h before fixed with PBS containing 4% methanol overnight at 4°C. Mouse anti-core mAb (1:100, Abcam #ab2740) and Alexa Fluor 488 goat anti-mouse IgG (H + L) antibody (1:1000, Life Technologies A-11029) were sequentially incubated with the sample for 1 h at 37°C, followed by PBS washing. Images were taken by DeltaVision Elite (Applied Precision, Issaquah, WA). Detailed protocol is the same as previously described49 (link).