Mouse anti cd81

To obtain whole-cell extracts, NSPCs were harvested in a RIPA buffer (320 mM sucrose, 50 mM TRIS pH 7.5, 1% Triton X-100, 10% glycerol) and 1% of inhibitor of proteases cocktail (Sigma-Aldrich), incubated on ice for 30 min and centrifuged for 12 min at 13,000 g. Protein amount was quantified by Bradford assay (BioRad, Hercules, CA, USA) and 10–20 μg of protein was subjected to SDS-polyacrylamide gel electrophoresis and transferred overnight onto a polyvinylidene difluoride membrane (Immobilon-PSQ, Millipore). Membranes were blocked in 5% nonfat dry milk and then incubated with the following primary antibodies: rabbit anti-HSP90 (1:300, Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-TSG101 (1:200, Santa Cruz Biotechnology, Dallas, TX, USA), goat anti-CD63 (1:1000, BD Biosciences, San Jose, CA, USA), mouse anti-CD81 (1:500, BD Biosciences, San Jose, CA, USA). Blots were washed, incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (1:10,000, Jackson Immunoresearch, West Grove, PA, USA), then washed again and finally incubated in lumilight-enhanced chemiluminescence substrate (BioRad) and exposed to Chemidoc (BioRad). Densitometric analysis on scanned blots was performed using the ImageLab program (BioRad).

A pcDNA3.1 vector carrying the TACSTD2 gene insert and a pcDNA3.1 vector carrying the siRNA-resistant TACSTD2 gene devoid of the 3’- and 5’-untraslated regions (UTR) (pTACSTD2) for rescue experiments were purchased from GenScript Corporation. siRNA for TACSTD2 gene silencing (siTACSTD2) targeting the 3’UTR of the TACSTD2 gene was performed using SMARTpool of 4 siRNAs (Dharmacon) (siTD2-A, 5’-GAGAAGAGGAGUUUGUUAAUU-3’; siTD2-B, 5’-ACAAGUAUCUGUAUGACAAUU-3’, siTD2-C, 5’-GCAAGUAACUGAAUCCAUUUU-3’, siTD2-D, 5’-GCACACACCAGGUUUAAUAUU-3’), which were transfected into parental and TACSTD2-overexpressing Huh 7.5 cells at 100nM final concentration using RNAiMAX (Invitrogen). The antibodies used include mouse anti-TACSTD2 (Santa Cruz), goat anti-TACSTD2, biotinylated goat anti-TACSTD2 and goat anti-E-cadherin, mouse IgG1 isotype control, mouse IgG2A isotype control, and normal goat IgG control (R&D systems), mouse anti-CLDN1 (Abnova), rabbit anti-CLDN1 and mouse anti-OCLN (Life Technologies), rabbit anti-OCLN (Abcam), rabbit anti-CD81 and mouse anti-ZO-1 (Thermo Scientific), mouse anti-SR-B1 (BD transduction laboratories), mouse anti-CD81 (BD Pharmingen), mouse anti-JAM-A (Hycult Biotech), mouse anti-HCV core (Anogen), rabbit anti-PKC substrate (Cell Signaling Technologies), and rabbit anti-alpha tubulin (Millipore). The HCV neutralizing mAb AR4A was a gift from Dr. Mansun Law.

Antibodies used in this study included anti-NS5A (Austral Biologicals), anti-Core (Affinity Bioreagents Inc.), anti-bromodeoxyuridine (Sigma-Aldrich), anti-EEA1 or -CD63 or -LAMP-1 (Santa Cruz Biotechnology), mouse anti-CD81 (BD Pharmingen), rabbit anti-CD81 (Santa Cruz Biotechnology), anti-E2 (GeneTex, Inc.), anti-calnexin (Assay designs/Stressgen), and anti-mouse or -rabbit colloidal gold conjugates (Jackson ImmunoResearch Inc.). Rabbit polyclonal Abs against Core (RR8) and NS4B (RR12) were described previously [15] (link). Cy3-conjugated primary Ab to β-tubulin and nonspecific normal mouse immunoglobulin G (IgG) were obtained from Sigma-Aldrich. Secondary Abs against mouse, rabbit and goat were purchased from Invitrogen Molecular Probes.
Reagents used were Nocodazole (Sigma-Aldrich), taxol (paclitaxel; Sigma-Aldrich), BODIPY 493/503 (Invitrogen Molecular Probes), 4',6-Diamidino-2-phenylindole (DAPI; Invitrogen Molecular Probes) and wheatgerm agglutinin (WGA) Alexa Fluor 647 conjugate (Invitrogen Molecular Probes).

Liver lysates (10 μg in total protein) and in vivo liver EVs (1 μg in total protein) were loaded onto SDS‐PAGE gels, and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membrane was blocked and incubated with horseradish peroxidase‐conjugated secondary antibodies, goat anti‐rat IgG, goat anti‐hamster IgG, goat anti‐mouse IgG, and goat anti‐rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immunoreactive bands were visualized using enhanced chemiluminescence substrate (Thermo Scientific, Hudson, NH, USA). Rat anti‐CD9, mouse anti‐CD81 and mouse anti‐GM130 antibodies were from BD Biosciences (BD Biosciences, San Jose, CA, USA). Rabbit anti‐histone H2B antibody was purchased from Upstate Biotechnology (Lake Placid, NY, USA).