Benzonase

The translated peptides were further validated by western blot. Transfected N2A Cells were harvested with 100 μl of RIPA buffer (25 mM Tris•HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) added with 1× PIC (Merck, #539131) and Benzonase (NovoProtein, #M046-01B) and incubated 10 min on ice. For detection of peptides from Brip1os, Miat, u-Rnf10 and DHFR, lysates were added with 5× SDS-PAGE Sample Buffer (GenStar, #E153) and denatured at 95°C for 5 min, and samples were loaded on 4–20% Mini-PROTEAN^® TGX™ Precast Protein Gel and transferred to a 0.2 μm NC membrane. For detection of the remaining peptides, lysates were added with 2× Novex Tricine SDS Sample Buffer (Invitrogen, #LC1676) and denatured at 85°C for 2 min, and samples were loaded on 16.5% GLASS Gel^® Tricine gel (WSHT, #TCH2001-16.5T) and transferred to a 0.1 μm NC membrane. The blot was carried out using Nano-Glo^® HiBiT Blotting System (Promega, #N4210) according to the manufacturer's instructions.

Cells were harvested and washed by PBS at 10,000 rpm at 4°C for 30 s. The cell pellet was resuspended in 1 ml of lysis buffer [137 mM NaCl, 20 mM tris-HCl (pH 8.0), 10% glycerol, 1% NP-40, 2 mM EDTA, and 1% protease inhibitor cocktail] on ice for 30 min and sonicated using a microtip at 35% amplitude for 1 s 10 times. The sonicated samples were then centrifuged at 12,000 rpm at 4°C for 15 min. Antibodies (1 to 2 μg) and protein A/G agarose beads were added to the supernatant lysate and incubated at 4°C overnight with a rotary shaker. For Benzonase treatment, 1-U Benzonase (M046-01A, Novoprotein) was added in the lysates. The immunoprecipitants were washed with wash buffer [20 mM tris-HCl (pH 8.0), 0.1% NP-40, 100 mM NaCl, and 1 mM EDTA] three times and eluted by boiling for 5 min with loading buffer. After centrifugation, the supernatant was subjected to Western blotting.

Benzonase was purchased from Novoprotein (Suzhou, China, #M056-01). Deoxycholic acid (#30970) and beta-mercaptoethanol (#M3148) were purchased from Sigma (St. Louis, MO, USA). Poloxamer 188 was purchased from BSAF (Germany, Art 50259527, #67056). Recombinant SARS-CoV-2 spike RBD recombinant protein was purchased from Sino biological (Beijing, China, #40592-V08H). The information of all flow cytometry antibodies is listed in supplementary materials (Supporting Information Table S1). The Milli-Q water (≥18 megohm.cm) water were used in all experiments for preparing all corresponding solutions. Specific pathogen-free (SPF) 6‒8-week-old female BALB/c mice were purchased from Dashuo Laboratory Animal Technology Co., Ltd. They were housed under SPF conditions, with 12-h light cycles and free access to regular chow diet and water, in the laboratory animal facilities at West China School of Pharmacy, Sichuan University. Animal studies were approved by the Committee on the Ethics of Animal Experiments of Sichuan University and conducted in compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Sichuan University Ethics Committee.