Ultracentrifuge tube

5-50% linear sucrose gradients were freshly prepared in an ultracentrifuge tube (Beckman) using an automated gradient maker (BioComp). Cells were pre-treated with 100 μg/ml Cycloheximide (CHX) (MCE) at 37 °C for 10 min, washed twice with ice-cold PBS containing 100 μg/ml CHX, and collected. Cells were then lysed in hypertonic buffer containing 20 mM Tris-HCl (pH7.4), 300 mM NaCl, and 10 mM MgCl₂, and 0.5% sodium deoxycholate, 1% Triton X-100, 1 mM DTT, 100 µg/ml CHX and 300 U/ml of RNase inhibitor were added. Cell debris was removed via centrifugation at 16,000 × g for 10 min at 4 °C. 500 µL of supernatant was then loaded into the sucrose gradients, followed by centrifugation at 4 °C and 36,000 rpm for 2 h (SW41Ti rotor, Beckman). Samples were then fractioned and analyzed with a Piston Gradient Fractionator (BioComp) and fraction collector (Gilson). 5 ng of polyadenylated synthetic luciferase mRNA (Promega) was added to each fraction for normalization. RNAs were extracted from each fraction and subjected to qRT-PCR analysis.

Sucrose density gradient analysis was performed at 4 °C. Cells (CFSC or primary rat HSC) were grown to near confluence in 10 cm dishes. After washing with ice-cold phosphate-buffered saline (PBS), the cells were scraped into 0.85 mL of sodium carbonate buffer (500 mM, pH 11.0). The cell pellets were homogenized in an ultrasonic disintegrator (model UP100H from Hielscher Ultrasound Technology, Teltow, Germany) with three 20-s bursts on ice. The homogenates were adjusted to 45% sucrose using 0.85 mL 90% sucrose in MES-buffered saline (25 mM MES, 150 mM NaCl, pH 6.5) and placed at the bottom of an ultracentrifuge tube (Beckman Coulter). Two solutions (1.7 mL each) of 35% and 5% sucrose were laid sequentially on the top of the 45% sucrose solution. After ultracentrifugation at 39,000 rpm in a Beckman Optima^TM L-70K centrifuge using a SW 40 Ti rotor for 20 h, 11 individual 0.5-mL fractions were collected from the top of the tubes, and 50 µL of each fraction were analyzed by Western blot (Section 2.13).

Fluorescent VLPs were made by cotransfection of pGAG‐mCherry plasmid (kind gift from Gummuluru lab) and SARS‐CoV‐2 S protein plasmid in a 1:1 ratio. Nonreplicating lentivirus pseudotyped with SARS‐CoV‐2 UK spike protein was created using the following plasmids, in a 1:1:1:2 ratio: pMDLg/pRRE, pRSV‐REV, SARS‐CoV‐2 UK Spike, and pLL7 GFP. The plasmids were transfected into LVMaxx using the LVMaxx Transfection kit. Each VLP was made in 60 ml cultures, and harvested after 48 h. The VLPs were purified using 25% sucrose (in 25 mM HEPES/130 mM NaCl) cushion spin protocol. Three milliliters of 25% sucrose solution was add to each Beckman Coulter ultracentrifuge tube, which then had 7 ml of cell culture supernatant gently layered on top. The tubes were then spun at 36,000 rpm for 2.5 h at 4°C. The sucrose/supernatant was then aspirated off, and 20 μl of 10% Sucrose solution was placed on top of the VLP pellet. After 24 h at 4°C, the VLPs were then aliquoted and stored at ‐80°C.

Detergent-resistant membranes (DRM) were produced from 15 × 10⁶ cells using Triton X-100 lysis and isolated by sucrose density gradient ultracentrifugation according to Jouannet et al. (2016) (link). Briefly, 15 × 10⁶ cells were lysed in 250 μL of lysis buffer (20 mM Tris pH 7.5, 1% Triton X-100, 150 mM NaCl), containing protease inhibitor cocktail for 30 min on ice. 200 μL lysate was then mixed with 800 μL of 60% sucrose (prepared in lysis buffer with detergent, supplemented with protease inhibitor), and placed on the bottom of 5mL ultracentrifuge tube (344,057, Beckman). The sample was overlaid with 2.5mL of 30% sucrose and 1mL of 2,5% sucrose (prepared in lysis buffer without detergent, supplemented with protease inhibitor) and centrifuged at 200,000 g for 16 h at 4°C in swing buckets of MLS-50 rotor using an Optima MAX-XP ultracentrifuge. After ultracentrifugation, 9 fractions of 0.5mL were collected from top (low sucrose concentration) to bottom of the tube (high sucrose concentration). Fractions were analyzed by immunoblot analysis.

Lipid-raft microdomain isolation was performed exactly as previously described [43 (link)]. Briefly, mice were euthanized and one hemisphere of the forebrain was homogenized by 30 strokes in a class B Dounce homogenizer in 2 mL of homogenization buffer (150 mM NaCl, 25 mM MES, and 1× protease inhibitor, pH 6.5). After five passages through a 26-gauage needle, the homogenate was centrifuged at 1000×g for 10 min to remove cell bodies. An equal amount of protein from the resultant supernatant was then incubated with Brij-58 (1% final volume) at 4 °C for 1 h. Sucrose (40% final concentration by volume) was then added to the sample. One milliliter of the sample was loaded into an ultracentrifuge tube (Beckman) and overlaid with 1.8 mL of 30% sucrose and 1.2 mL of 5% sucrose (both resuspended in homogenization buffer). The gradients were centrifuged overnight (16 h) at 4 °C at 175,587×g. A total of 10 fractions (400 μL/fraction) were isolated using a pipette starting from the top, and the pellet (fraction 11) was resuspended in 400 μL of homogenization buffer. Protein readings were analyzed for each fraction. For Western blot analysis, the same amount of protein was loaded into each lane. The lipid raft marker flotillin-1 was used to identify the fractions that represent the lipid raft microdomain.