Goat anti mouse sc 2005

Proteins extracted from cells or concentrated from elutriants were analyzed with SDS-PAGE, the proteins on the membrane were probed with anti-HCV Core (C7-50, Abcam Ltd.), anti-NS3 (H23, Abcam Ltd.), anti-hA3G (ab75560, Abcam Ltd.), anti-HA (6E2, Cell Signaling Biotechnology Inc.), or anti-HA-tag [HRP] (A00169, GenScript.) antibody, respectively, with anti-Actin antibody (TA-09, ZSGB-BIO, China) served as the control. After washing with TBST, the membrane was incubated with goat anti-mouse (sc-2005, Santa Cruz Biotechnology Inc.) or goat anti-rabbit (sc-2004, Santa Cruz Biotechnology Inc.) secondary antibody, respectively. Protein signals were visualized and captured using Immobilon Western Chemiluminescent HRP Substrate ECL working solution (Millipore Inc.) with ChemiDo XRS gel imager system (Bio-Rad, CA).

Antiretroviral drugs TDF, FTC (Gilead Sciences, Foster City, CA, USA), and DTG (ViiV Healthcare, Research Triangle Park, NC, USA) were used. NAC (A7250) and (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO; SML0737) were purchased from Sigma-Aldrich, St. Louis, MO, USA, and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL; sc-200825) was purchased from Santa Cruz Biotechnology, Dallas, TX, USA. Antibody resources: beclin 1 (BECN1; sc-11427) was purchased from Santa Cruz Biotechnology, Dallas, TX, USA. Lysosome associated membrane protein 2 (LAMP2; NB300-591), microtubule-associated protein 1 light chain 3 beta (MAP1LC3B; NB100-2220), and integrin subunit alpha M (ITGAM; NB110-89474) were purchased from Novus Biological Company, Centennial, CO, USA. Cathepsin D (CTSD; ab75852) and caspase 3 (CASP3; ab13585) were purchased from Abcam, Cambridge, MA, USA. Galectin 3 (GAL3) (A3A12) was purchased from Invitrogen. Sequestosome 1 (SQSTM1; PM045) was purchased from MBL International, Woburn, MA, USA, and allograft inflammatory factor 1 (AIF1; 019-19741) was purchased from Wako Pure Chemicals Industries, Chuo-ku, Osaka, Japan. Goat anti-rabbit (sc-2004) and goat anti-mouse (sc-2005) were purchased from Santa Cruz Biotechnology, Dallas, TX, USA.

Total protein extract was obtained by homogenizing tissue in RIPA buffer (PBS, 0.5% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 5.5% β-glycerophosphate, 1 mM dithiothreitol and complete protease and phosphatase inhibitors (Roche Diagnostics, Vilvoorde, Belgium)). The total protein yield was determined using Bradford reagent (Biorad, Temse, Belgium) and 30 µg protein was fractionated by SDS-PAGE. Protein lysates from multiple mice per group were pooled and transferred to a nitrocellulose membrane which was subsequently blocked for 1 h with 5% BSA, incubated overnight at 4 °C with primary antibodies in blocking buffer (Table S2), followed by 1 h incubation at room temperature with horse radish peroxidase-conjugated secondary antibodies (goat-anti rabbit sc2004, Santa Cruz, Heidelberg, Germany or goat anti-mouse sc2005, Santa Cruz). Clarity western ECL substrate (Biorad, Temse, Belgium) was used to visualize the proteins. GAPDH and β-tubulin were used as loading control proteins or total protein load was used for normalization.

Compounds sofosbuvir (PSI-7977), telaprevir (VX-950), and simeprevir were purchased from MedChemExpress (Princeton, NJ). The monoantibody (mAb) to HCV Core (ab2740) or NS3 (ab13830) was from Abcam, Co. Ltd. The mAb to beta-actin (TA-09) was from Beijing ZSJQ-BIO Co. Ltd. Goat anti-mouse (sc-2005) and goat anti-rabbit (sc-2004) secondary antibodies were from Santa Cruz Biotechnology Inc.