Normal rabbit igg ac antibody

Total cellular proteins from Huh7.0, JFH1-infected Huh7.0, and HCV FL replicon cells were extracted using RIPA buffer (cat. #BP-115, Boston Bioproducts) as previously described. Proteins resolution was done on an 8–15% gel SDS-PAGE denaturing gel. Resolved proteins in acrylamide gels were transferred onto Polyvinylidene fluoride (PVDF) membranes and membranes were blocked in PBS containing 5% dry milk for 1h, then probed with specific primary antibodies in the recommended dilutions at 4°C for overnight. The following primary antibodies were used: anti-HCV NS3 (Abcam cat. #ab13830 and cat. #ab65407); anti-HCV NS5A (Abcam cat. # ab13833 and BioFront cat. # HCV-2F6); anti-HSP90 (Abcam cat. # ab13492); anti-FLAG (Abcam cat. #ab1162); anti-CD63 (Abcam cat. #ab8219 and Santa Cruz Biotechnology cat. #sc-15363); RTN3 antibody (Abcam cat. Ab68328 and Thermofisher cat. # PA5-53360); TSG101 (Abcam cat. # ab125011); normal rabbit IgG-AC antibody (Santa Cruz Biotechnology cat. # sc-2345); anti-beta actin [Ac-15] (Abcam, cat. #ab6276). Appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology cat. # sc-2004 and sc-2005) and clarity TM Western ECL blotting substrate (Bio-Rad) was used for visualization with the ChemiDoc XRS+ system (Bio-Rad, California, United States).

Western blots were performed using the following established protocols. Briefly, proteins were resolved on 10% SDS-PAGE gels. After electrophoresis resolved proteins were transferred onto nitrocellulose membranes. Following protein transfer, membranes were blocked for 1 hour in PBS containing 5% non-fat dry milk and 0.1% Tween-20. Blots were then incubated overnight with primary antibody at 4°C. The following primary antibodies were used: anti-HCV NS3 (Abcam cat. #ab13830); anti-HSP90 (Cell Signaling cat. #4874); anti-CD63 (Abcam cat. #ab8219 used for western blotting and Santa Cruz Biotechnology cat. #sc-15363 used for exosomes purification); anti-Ago2 (Sigma cat. #SAB4200274); anti-CD81 (Santa Cruz Biotechnology cat. #sc-23962), normal rabbit IgG-AC antibody (Santa Cruz Biotechnology cat. # sc-2345); anti-beta actin [Ac-15] (Abcam, cat. #ab6276). The membranes were then incubated for 1 hour with horseradish peroxidase-conjugated secondary antibodies (dilution 1∶10,000) that included: goat anti-mouse IgG-HRP (Santa Cruz Biotechnology cat. #sc-2005); goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology cat. #sc-2004). Finally, the proteins were visualized with the Clarity Western ECL substrate (BioRad, cat. #170-5061) chemiluminescence system according to the manufacturer's protocol using the Fujifilm LAS-4000 luminescent image analyzer.