Tla 110

First, 164K pellets collected by differential centrifugation from 8–15 × 10⁷ cells were resuspended in 1.1 mL of STE buffer (0.25 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4) and transferred to the tube of SW60Ti rotor (Beckman) and mixed 1:1 with 60% (wt/vol) stock solution of iodixanol (Optiprep medium, Sigma). STE buffer diluted iodixanol solutions, 1 mL of 20% iodixanol, and 0.8 mL of 10% iodixanol were layered sequentially on the top and the tube centrifuged at 350,000× g and 4 °C for 1 h (brake off). Eight fractions of 480 μL were collected from the top of the tube. For Western blot and CCA in the downstream analyses, 320 μL and 160 μL (respectively) from each 480 μL fraction was diluted with 2 mL and 240 μL PBS (respectively) and centrifuged at 164,000× g for 30 min in TLA110 and TLA 120.1 rotors (Beckman; respectively). For Western blot and immuno-isolation in the downstream analyses, each fraction was split into two 240 μL portions, diluted with 2 mL PBS for Western blot and 560 μL PBS for immuno-isolation, followed by centrifugation in TLA110 and TLA 120.2 rotors (Beckman), respectively. The pellets were resuspended in PBS for immuno-isolation, radioimmunoprecipitation assay (RIPA) lysis buffer (Cell Signaling) for Western blot, or Quick C-Circle Preparation (QCP) lysis buffer [16 (link)] for CCA, and stored at −20 °C.

Virus particles were purified from 30 g frozen mycelium according to the method of Lot et al. (1972) [16 ]. The pellet was resuspended in 150 μL borate buffer (5 mM boric acid, 1.475 mM sodium tetraborate, 0.5 mM EDTA) and then placed on a discontinuous sucrose density gradient (10% to 40% (w/v) sucrose in PBS) for ultracentrifugation at 70,000× g for 2 h at 4 °C in a swinging-bucket rotor (Beckman Coulter MLS-50). VLPs were collected from the ~35% sucrose layer, dialyzed against PBS (16 h at 4 °C), and pelleted by ultracentrifugation at 144,000× g for 2 h at 4 °C in a fixed-angle rotor (Beckman Coulter TLA-110). The VLP-containing pellet was resuspended in 30 μL PBS and analyzed by transmission electron microscopy. Purified virus particles were analyzed with a JEM-1400 Flash transmission electron microscope (JEOL) to identify the morphological characteristics of the particles. We used the method described in [7 (link)] to obtain the negatively stained samples, which were systematically screened at 30,000× magnification to localize the presence of the virus particles on the grid. Afterwards, the particles were recorded at 60,000× magnification with a 16 MP Matataki Flash scientific complementary metal–oxide–semiconductor (sCMOS) camera (JEOL).

Protein extracts from lumbar spinal cords or NSC-34 cells were prepared by homogenization in lysis buffer containing 50 mM Tris HCl pH7.5, 150 mM NaCl, 2 mM EDTA, 1 % Triton X100, protease inhibitors (Complete EDTA-free, Roche). 50 μg protein were subjected to SDS-PAGE and blotted on Immobilon membranes (Millipore) which were processed by standard methods and revealed with Immobilon Western kits (Millipore). Band intensities were quantified by TotalLabQuant software.
Crude fractionation of membranes from lumbar spinal cord was performed after tissue freezing (−80 °C), thawing and homogenization in 50 mM HEPES, pH 7.4, 250 mM sucrose, 1 mM Mg-acetate and protease inhibitors (Complete EDTA-free, Roche). Lysates were homogenized using a Dounce homogenizer (15 passes) and centrifuged at 1.000 g for 10 min. The postnuclear supernatant was centrifuged at 10.000 g for 30 min at 4 °C yielding a P10 pellet and the supernatant was centrifuged at 100.000 g (Beckman TLA-110) for 1 h at 4 °C yielding an S100 supernatant and a P100 pellet.