0.45 m nitrocellulose membrane

Total proteins were extracted with protein extraction buffer (Agrisera, Vannas, Sweeden) supplemented with 5 mM DTT and the cocktail of protease inhibitors to prevent protein degradation. An amount of 10 µg of proteins were separated on 15% acrylamide SDS–PAGE and transferred to 0.45 µm nitrocellulose membrane (Amersham, Buckinghamshire, U.K., catalogue number 10600003). All proteins apart from WHY1 were detected with rabbit polyclonal primary antibody (Agrisera) and secondary HRP-linked anti-rabbit (1 : 10000, Agrisera catalogue number AS09 602).
For immunological detection of WHY1, the antibodies were directed toward the synthetic peptide of recombinant HvWhy1 protein (PRQYDWARKQVF) in rabbits and antibodies were affinity-purified (Generon, Slough, U.K.). The specificity of immunodetection was validated using pre-immune sera.

Protein samples were extracted from cells and mitochondria, respectively, using RIPA lysis buffer (Chromotek) supplemented with protease inhibitors. Protein samples were diluted in LDS sample buffer (4×, Invitrogen) supplemented with sample reducing agent (10×, Invitrogen). Samples were heated at 95̊C for 5 min before loading on a 4–12% gradient Bis-Tris gel (Invitrogen). MOPS SDS running buffer (Invitrogen) was used and supplied with antioxidants (Invitrogen). The gel was soaked in running buffer and run at 160 V for 75 min. The gel was transferred to a 0.45 µm nitrocellulose membrane (Cytiva) and protein at 30 V for 150 min, 4̊C. The membrane was blocked for 1 h in 5% skimmed milk (Sigma) in PBS-Tween 20 (0.1%, Sigma). The membrane was probed for anti-GFP (ab183734, abcam), anti-mCherry (1C51, ab125096, abcam), anti-VDAC1 (ab15895, abcam) and anti-α tubulin (DM1α, T6199, Sigma), diluted 1 : 10 000, 1 : 3000, 1 : 1500, 1 : 1500, respectively, in 5% milk (PBS-Tween). Primary antibody signal was detected using goat anti-mouse IgG conjugated to IRDye 680RD (ab216776, abcam) and goat anti-rabbit IgG conjugated to IRDye 800CW (ab216773, abcam) secondary antibodies, diluted 1 : 15 000, imaged with an Odyssey CLx imaging system (LI-CO Biosciences).

Cell lysates were prepared using ice-cold cell-lysis buffer (50 mmol/L HEPES, pH 7.5; 150 mmol/L NaCl; 1% triton X-100; 2% aprotinin; 2 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 8.0; 50 mmol/L sodium fluoride; 10 mmol/L sodium pyrophosphate; 10% glycerol; 1 mmol/L sodium vanadate; and 2 mmol/L Pefabloc SC) for 30 min. Subsequently, samples were centrifuged at 13,000 rpm for 10 min in a table top centrifuge. Supernatants were transferred to new tubes and stored at −80 °C till use. A total of 10 µL of virus sample was mixed with loading dye. SDS-PAGE of proteins was performed on a 10% polyacrylamide gel (for detection of RSV F and VSV N: heated loading buffer without β-Mercaptoethanol added to samples; for detection of LCMV GP: samples boiled in loading buffer with β-Mercaptoethanol). Subsequently, proteins were transferred to 0.45 µm nitrocellulose membranes (Whatman, Dassel, Germany) and membranes were blocked with MPBST (PBS containing 5% skim milk and 0.1% Tween-20). Proteins were detected using primary antibodies (RSV F (18F12) [36 (link)], VSV N (Kerafast, Boston, USA), β-actin (clone AC-74, Sigma-Aldrich, St. Louis, MO, USA)) or hybridoma supernatant (LCMV GP (KL25)) and appropriate peroxidase-conjugated secondary antibodies.

Samples for immunoblotting were collected from 293T cells expressing VSV-L. We used this replication-supporting cell line for the expression of sufficient fusion protein (GFP-3CLpro-L) in the non-active condition (+ PI) of VSV-3CLpro-Off. SDS-PAGEs of protein lysates were performed under reducing conditions, on an 8% polyacrylamide gel for VSV-GFP and VSV-3CLpro-Off constructs. Gels were run for 90 min at 100 Volt. Proteins were transferred to 0.45-µm nitrocellulose membranes (Whatman, Dassel, Germany) by using a tank blotting system (Bio-Rad, Hercules, CA, US). The blotting time was 80 min. Blotting buffer contained 15 % methanol. The membranes were blocked overnight with 1x PBS containing 5% skim milk and 0.1% Tween 20 (PBSTM). GFP was stained by a mouse antibody (clones 7.1 and 13.1; Roche, Basel, Switzerland) diluted 1:1000 in PBSTM and a rabbit GFP/YFP antibody produced by Stephan Geley (conc.: 0.3 mg/mL) diluted in 1:2000 in PBSTM. β-Actin was stained with a monoclonal mouse anti-Actin antibody, A5441-.5 ML from SIGMA diluted 1:1000 in PBSTM. Blots were not stripped prior to β-Actin staining. PageRuler^TM Prestained Protein ladder 26616 (ThermoFisher, Massachusetts, USA) was used as marker.

To characterize membrane orientation of PalmGRET and GRET in sEVs, sEVs isolated from 0.22 µm filtered CM were serially diluted to the following concentrations: 31.25, 62.5, 125, 250, 500, 1000, and 2000 ng in 5 µL. Cell lysates were diluted to 2000 ng in 5 µL as positive controls. 5 µL of diluted sEVs, cell lysates, and double‐filtered PBS were dotted onto 0.45 µm nitrocellulose membranes (Amersham Bioscience) and blocked in 10% BSA supplemented PBS overnight at 4 °C. The membranes were immunoprobed with anti‐GFP antibody (GeneTex) diluted in 5% BSA in PBS or PBST overnight at 4 °C, wash three times in PBS or PBST for 30 min each, and incubated with HRP‐conjugated secondary antibodies for 1 h at room temperature followed by another 3 washes. The membranes were developed by chemiluminescence using ECL Select Western Blotting Detection Reagent (Amersham Bioscience) and MultiGel‐21 Imaging System (Topbio). Antibody dilutions and hosts are provided in Table S2 (Supporting Information).