Mouse anti human cd81

Vesicles recovered from conditioned medium from 20 × 10⁶ cells (2 K, 10 K and 100 K pellets), equivalent volumes of cell-free medium (2 K, 10 K and 100 K pellets) or 15 μl of the iodixanol gradient fractions were loaded on 4–15% or 10% Mini‐Protean® TGX Stain‐Free™ gels (Bio‐Rad), under non‐reducing conditions. Transferred membranes (Immun‐Blot PVDF Bio‐Rad) were developed (e.g. using Clarity western ECL substrate (Bio-Rad) or SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo Fisher) and a ChemiDoc Touch imager (Bio‐Rad) or by standard film exposure). Intensity of the bands was quantified using ImageLab Software (Bio‐Rad). Antibodies for WB were mouse anti‐human CD63 (clone H5C6, BD Bioscience #557305), mouse anti-human CD81 (Santa Cruz sc-23692), goat anti-AChE (ab31276, Abcam) and anti-HIV-1 p24 Monoclonal (183-H12-5C, NIH AIDS reagent program) or Anti-HIV1 p55 + p24 + p17 (ab63917, Abcam). Secondary antibodies included HRP-conjugated goat anti-rabbit IgG (Santa Cruz, sc-2004) and HRP-conjugated m-IgG-k BP (Santa Cruz, sc-516102) (JHU), or (CT lab) HRP-conjugated anti-mouse (Jackson Immunoresearch, 115–035-146) and anti-goat (Jackson, 705–035-147).

Cells were fixed with ice-cold methanol for 10 min. After washing with PBS-0.05% Tween 20 (PBST) and blocking in 0.5% BSA in PBS for 1 hr, cells were incubated with primary antibodies overnight at 4°C. Primary antibodies used were rabbit anti-human Oct4 (Abcam), mouse anti-human Albumin (Sigma-Aldrich), rabbit anti-human HNF4α (Santa Cruz Biotechnology), mouse anti-human E-cadherin (Abcam), mouse anti-human Occludin (Invitrogen), mouse anti-human CD81 (Santa Cruz Biotechnology), mouse anti-human claudin-1 (Invitrogen), mouse anti-human SR-BI (BD), mouse anti-NS5A (9E10, a kind gift from Charles M. Rice, Center for the Study of Hepatitis C, The Rockefeller University, New York), rabbit anti-HCV core serum (R308, a kind gift from John McLauchlan). Secondary antibodies were Alexa Fluor 488 donkey anti-mouse (Molecular Probes), Alexa Fluor 488 donkey anti-rabbit (Probe molecular), and Alexa Fluor 594 donkey anti-rabbit (Molecular Probes) conjugates. Details of working dilution of each antibody are provided in Table S1. Cells were counterstained with DAPI (Sigma-Aldrich), and the pictures were captured by microscope of Zeiss Axio Observer.