HEK293FT cells were transiently transfected with equal amounts of Spike or ACE2 vectors using PEI. Cells were collected 18–24 hr post-transfection with TrypLE (Thermo), washed twice with PBS (Caisson Labs), and lysed with TNE buffer (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) supplemented with protease inhibitor cocktail (Bimake B14001) for 1 hr on a rotator at 4°C. Cells lysates were spun for 10 min at 10,000 g at 4°C, and protein concentration was determined using the BCA assay (Thermo 23227). Whole-cell lysates (10 μg protein per sample) or 5 μL of 100× concentrated Spike-pseudotyped lentiviral particles were denatured in Tris–glycine sodium dodecyl sulfate (SDS) sample buffer (Thermo LC2676) and loaded on a Novex 4–12% Tris–glycine gel (Thermo XP04122BOX). PageRuler pre-stained protein ladder (Thermo 26616) was used to determine the protein size. The gel was run in 1× Tris–glycine–SDS buffer (IBI Scientific IBI01160) for about 120 min at 120 V. Protein transfer was performed using nitrocellulose membrane (BioRad 1620112) using prechilled 1× Tris–glycine transfer buffer (Fisher LC3675) with 20% methanol for 100 min at 100 V. Membranes were blocked with 5% skim milk dissolved in PBST (1× PBS + 1% Tween 20) at room temperature for 1 hr. Primary antibody incubations were performed overnight at 4°C using the following antibodies: rabbit anti-GAPDH 14C10 (RRID: AB_561053) (0.1 μg/mL, Cell Signaling 2118S), rabbit p24 monoclonal antibody clone 002, which recognizes lentiviral capsid protein (1 μg/mL, Sino Biological 11695-V08E), and mouse anti-rhodopsin antibody clone 1D4 (RRID:AB_10010560) (1 μg/mL, Novus NBP1-47602), which recognizes the C9-tag added to the Spike proteins. Following the primary antibody, the blots were incubated with IRDye 680RD donkey anti-rabbit (AB_10954442) (0.2 μg/mL, LI-COR 926–68073) or with IRDye 800CW donkey anti-mouse (AB_621847) (0.2 μg/mL, LI-COR 926–32212) for 1 hr at room temperature. The blots were imaged using Odyssey CLx (LI-COR). Band intensity quantification was performed by first converting Odyssey multichannel TIFFs into 16-bit grayscale image (Fiji) and the then selecting lanes and bands in ImageLab 6.1 (BioRad). In ImageLab, background subtraction was applied uniformly across all lanes on the same gel.

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