Alliance q9 mini instrument

Cells were lysed in the Cell Lytic M buffer (Merck/Sigma, Darmstadt, Germany) supplemented with the cOmplete mini protease inhibitor cocktail (Roche). Total protein concentration was measured at 280 nm using the DS-11 Spectrophotometer (DeNovix, Wilmington, DE, USA). Cell lysates were resolved by 8% or 10% SDS-PAGE and transferred to PVDF membrane (Merck Millipore, Burlington, MA, USA), which was subsequently blocked with 5% bovine serum albumin (BSA, Merck/Sigma, Darmstadt, Germany). Primary antibodies were incubated for one hour at room temperature (RT). Goat B1B2 anti-PRV IE180 polyclonal antibody was used at a dilution of 1:5000 (from Dr. Hanns-Joachim Rziha). Mouse anti-PRV gE mAb was diluted at 1:2000. Mouse anti-β-actin monoclonal antibody (Novus Biologicals) was diluted at 1:5000. This was followed by HRP-conjugated secondary antibodies for 1 h at RT. The signal was detected by chemiluminescence using the Clarity Max Western ECL Substrate (Bio-Rad, Hercules, CA, USA) and the Alliance Q9 Mini instrument (Uvitec, Cambridge, UK). Densitometric analysis was performed using the Uvi Band software (Uvitec).

Proteins were isolated with RIPA buffer and transferred onto a 0.2 µm nitrocellulose membrane as described previously [40 (link)]. The membranes were blocked with 5% nonfat dry milk and stained with primary antibodies: anti-CD63 (Abcam, Cambridge, UK), calnexin (Cell signaling technology, Beverly, MA, USA) and β-actin (Cell signaling technologies). Membranes were also stained with amido black as a loading control. Afterwards, the membranes were stained with peroxidase-conjugated secondary antibody (Cell signaling technology) and visualized with the following chemiluminescent systems: Western Lightning^® Plus ECL (Perkin Elmer, Waltham, MA, USA) for cellular proteins and SuperSignal™ West Atto (Thermo Scientific, Waltham, MA, USA) for exosomal proteins. Images were obtained using an Alliance Q9 mini instrument (UVitec, Cambridge, UK).