Gm130

Drosophila S2 cells were plated on ibidi dishes (Planegg/Martinsried, Germany) or coverslips were fixed with 3% paraformaldehyde solution (pH 7.3) at 21°C for 10 min. In some cases, fixed cells were subsequently permeabilised by treatment with 0.1% Triton X-100 at 21°C for 10 min. In cases where antigen retrieval was required, proteins were denatured by the treatment of fixed and permeabilised cells with 6 M guanidinium hydrochloride (GdnHCl) at 21°C for 7 min. Cells were subsequently rinsed with either PBS in the case of non-detergent-treated cells or in the case of detergent-treated samples, with 0.01% Triton X-100 in PBS (PBST). Washed cells were incubated with mouse monoclonal anti-PrP antibody 4H11 [55 (link)] and the Golgi-specific rabbit polyclonal antibody GM130 (Abcam, Cambridge, U.K.) at 37°C for 60 min. After three washing steps in PBS or PBST, as appropriate, cells were incubated with Alexa Fluor-488-conjugated goat anti-mouse IgG (Invitrogen, Karlsruhe, Germany) or Cy3-conjugated goat anti-rabbit IgG (Dianova, Hamburg, Germany) at 21°C for 60 min. Nuclei were stained with Hoechst DNA staining dye (Sigma, Taufkirchen, Germany). Confocal laser scanning microscopy was performed on an LSM 700 laser scanning microscope (Zeiss, Göttingen, Germany).

Immunofluorescence analysis (IFA) was performed as described previously^{20 (link)}. Briefly, HeLa cells (2 × 10⁴ cells/well) grown on glass coverslips treated with or without specific pharmacological agents and CARDS toxin (140 pmol) or C-CARDS (70 pmol) were fixed in 2% paraformaldehyde, permeabilized with 0.1% Triton-X-100 and blocked with 1% normal goat serum (NGS; Gibco). Then, cells were washed with 0.2% NGS and treated with rabbit polyclonal anti-CARDS toxin (1:1000) as indicated previously and incubated with secondary goat polyclonal anti-rabbit antibody (1:1000 dilution) labeled with Alexa Fluor 555 (Invitrogen) for 1 h. Cellular F-Actin was stained with Alexa Fluor 488-conjugated phalloidin (Invitrogen). For co-IFA studies, cells were incubated with GM130 (1:1000, Abcam) or ERGIC (1:100; Santa Cruz) monoclonal antibodies in PBS with 0.2% NGS in PBS for 1 h. Cells were washed with PBS containing 0.2% NGS and incubated with secondary antibodies (Alexa Fluor 488 goat anti-mouse, 1:500) in PBS with 0.2% NGS for 1 h at RT. Individual samples were mounted in medium containing 4’,6-diamidino-2-phenylindole (DAPI; Vector Laboratories;), and images were acquired using a Carl-Zeiss immunofluorescence microscope, and Z sections were prepared using AxioVision deconvolution software and enhanced in Adobe Photoshop.

Primary antibodies used for western blotting are listed below: Tubulin (mouse, 1:2000, Developmental Studies Hybridoma Bank), ß-Actin (mouse, 1:2000, Santa Cruz), Rh1 (mouse, 1:2000, Developmental Studies Hybridoma Bank), TRPL (rabbit, 1:500, Fisher, Waltham, USA) [30 (link)], TRP (rabbit, 1:2000) [29 (link)], INAD(rat, 1:2000) [29 (link)]. The TRP and INAD antibodies were a gift form Dr. C. Montell. Secondary antibodies were bought from LI-COR Biosciences, which included IRDye 680 goat anti-mouse-IgG, IRDye 800 goat anti-rabbit-IgG, and IRDye 800 goat anti-rat-IgG antibodies (1:10000).
Primary antibodies used for staining are listed below: Rh1 (mouse, 1:200, Developmental Studies Hybridoma Bank), Cnx99A (mouse, 1:20, Developmental Studies Hybridoma Bank), GM130 (rabbit, 1:200, #ab30637, Abcam) GFP (rabbit, 1:200, Invitrogen), RFP (rat, 1:200, ChromoTek), EMC3 (rabbit, #ab175537, Abcam), NaK ATPase (mouse, #NA163540, Thermo Scientific), M-Opsin opsin (AB5405, Millipore, MA, USA), Rho 1D4 monoclonal antibody against rhodopsin was a gift from Dr. Robert Molday, University of British Columbia, Canada. Secondary antibodies were bought from Invitrogen (anti mouse, rabbit or rat IgG labeled with Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, or Alexa Fluor 488), 1:500 dilution.

Natural killer-Exo were treated with radioimmunoprecipitation assay buffer and vortexed three times for 10 min each to promote lysis. The lysates were centrifuged at 13,200 × g for 20 min and the supernatant was collected. Proteins (50 µg) present in the supernatant were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by transfer to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking, the membranes were incubated overnight at 4°C with primary antibodies against CD63, Alix, GM130, calnexin, perforin, and FasL (Abcam, Cambridge, UK). The membranes were washed three times for 10 min each in washing buffer (Tris-buffered saline and Tween 20) with shaking and then incubated with secondary antibodies for 1 h at room temperature. The membranes were washed again for 10 min three times, and the bands obtained were visualized using an enhanced chemiluminescence detection kit (Amersham plc, Amersham, UK).

L1CAM-positive NDE cargo proteins were quantified by using the human-specific ELISAs for Aβ_1–42 (Anogen, Ontario, CA), Aβ_1–40 (Anogen, Ontario, CA) and ExoELISA CD63 Kit (System Biosciences, Inc., Mountain View, CA) in duplicate with verification of bicinchoninic acid (BCA) reagent-based protein quantitation (Thermo Scientific, Inc.) to normalize the relative values for each sample.
L1CAM-positive plasma NDEs were characterized based on size and shape using transmission electron microscopy (TEM). The degree of purity was verified by western blot with positive exosomal marker CD63 (Abcam, Cambridge, MA, USA) and Tsg101 (Abcam, Cambridge, MA, USA) and negative exosomal marker GM130 (Abcam, Cambridge, MA, USA). The size of the samples was directly determined by NTA using a NanoSight LM10 microscope (NanoSight Ltd., Salisbury, UK).

Anti-SARS-CoV-2 Spike antibody, anti-nucleocapsid antibody, and anti-nucleoprotein antibody were from ProSci, Inc. (Poway, CA, USA). Anti-SARS-CoV-2 anti-Membrane (M) glycoprotein antibody was a custom synthesized rabbit polyclonal antibody to the peptide: KLNDTHSSSSDNIALLVQ (Thermo Fisher Scientific/Invitrogen, Waltham, MA, USA). Anti-TGN46, giantin, GM130, and calnexin were from Abcam (Cambridge, UK). Anti-TGN46 from Thermo Fisher Scientific/Invitrogen, Waltham, MA, USA was used in some experiments. Anti-Strep tag was from Sigma. Monoclonal antibody SCICONS J2 to dsRNA was from English and Scientific Consulting, Kft, Hungary. Secondary antibodies, anti-mouse Ig or anti-rabbit Ig, were Fab’-fragments conjugated to DyLight₄₈₈ or DyLight₅₉₄ or horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA, USA). All primary antibodies and fluorescent secondary antibodies were used at a dilution of 1:200. Anti-horseradish peroxidase secondary antibody was used at 1:100.
Helix pomatia agglutinin (HPA) lectin conjugated to AlexaFluor 488 was from Invitrogen and used at a final concentration of 5 μg/mL. Vital staining with C6-NBD-ceramide (Thermo Fisher Scientific/ Invitrogen, Waltham, MA, USA) was performed as previously described [15 (link)].