Iblot transfer system

Protein from neurospheres and isolated EVs was extracted using 1xRIPA lysis buffer (EMD Millipore; Reference # 20–188), with addition of Halt protease & phosphatase inhibitor cocktail (Thermo Fisher Scientific; Product # 78442). Extracted protein concentration was determined using Pierce BCA protein assay kit (Thermo Fisher Scientific; Catalog # 23225). 25ug of protein was size-fractionated on a 4–12% Bis-Tris Gel, ran at 150V for 60 minutes, and blotted to a PVDF membrane using iBlot transfer system (Thermo Fisher Scientific; Catalog # IB301001). Subsequently, the membrane was blocked for one hour with 2.5% goat serum and 5% nonfat dry milk in Tris-buffered saline that contains 0.1% Tween-20 (TTBS), then incubated overnight with polyclonal rabbit anti-CD63 antibodies diluted 1:1000 (System Biosciences; Catalog # EXOAB-CD63A-1). CD63 is an established surface marker for EVs. The blot was washed and incubated with an HRP-conjugated goat anti-rabbit IgG (Invitrogen) at dilution 1:1000 for one hour, then developed using PerkinElmer Western Lightning Plus-ECL (PerkinElmer; Catalog # NEL103001EA) and visualized using a CCD camera (Fluorchem Q, Alpha Innotech).

CHO cells were cultured and stably co-transfected with full-length pCAGGS-gp350/220 and pCI-puro vector containing a puromycin resistance gene. Forty-eight h post-transfection, DMEM media containing 10 μg/ml of puromycin was added to enrich for cells expressing gp350/220 protein. Puromycin-resistant clones were expanded, followed by flow cytometry sorting using m72A1 to a purity >90%. EBV gp350-positive CHO cells were harvested and lysed in radioimmunoprecipitation assay buffer (RIPA) followed by centrifugation at 15,000 g for 15 min on a benchtop centrifuge. The lysate was collected and heated at 95°C for 10 min in SDS sample buffer containing β-mercaptoethanol, then separated using SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane using an iBlot™ Transfer System (Thermo Fisher Scientific) followed by incubation in blocking buffer (1% BSA; 20 mM Tris–HCl, pH 7.5; 137 mM NaCl; and 0.1%Tween-20 [TBST]) for 1 h. The blots were incubated in TBST containing purified anti-gp350 antibodies (1:50) overnight at 4°C. After three washes with TBST, the blots were incubated with HRP-conjugated goat anti-mouse (1:2,000) in TBST for 1 h. After three washes, the antibody-protein complexes were detected using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare). All experiments were independently repeated three times.