Page gel silver staining kit

To express the prefusion Spike (S) ectodomain, a gene encoding the residues 1–1208 of 2019-nCoV S (GenBank: MN908947) with proline substitutions at residues 986 and 987, a “GSAS (amino acid sequence)” substitution at the furin cleavage site (residues 682–685), a C-terminal T4 fibritin trimerization motif, and an 8XHisTag were synthesized and cloned into the pcDNA3.1 vector. The plasmid was transfected to 293T cells and the recombinant S protein trimers were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography (QIAGEN, Hilden, Germany), followed by size exclusion to further purify the trimers. The trimeric Spike protein was dissolved in a Tris-glycine native sample buffer (Thermo Fisher, Waltham, MA, USA), and separated by a 3%–8% Tris-acetate protein gel under a non-reducing condition. The trimeric Spike protein was visualized by a PAGE gel silver staining kit (Solarbio, Beijing, China). The monomeric Spike protein was produced as a secreted form by BTI-Tn-5B1-4 insect cells as described by the authors of [55 (link)].

Biotin-labelled control (antisense) and circCRIM1 (sense) probes (Supplementary Table S1) were synthesized by TSINGKE. RNA pull-down assays were conducted as described previously [18 (link)]. The bound proteins in the pull-down samples were detected using Western blotting or mass spectrometry. Silver staining was performed according to the manufacturer’s instructions of the PAGE Gel Silver Staining Kit (Solarbio, Beijing, China). Mass spectrometry analysis was accomplished by Novogene (Tianjin, China) using an EASY-nLCTM 1200 UHPLC system (Thermo Fisher Scientific) coupled with a Q ExactiveTM series mass spectrometer (Thermo Fisher Scientific). Furthermore, protein identification and quantification were performed by Proteome Discoverer software (version 2.2, ThermoFisher Scientific).

PHA-L (L2769) was purchased from Sigma (Saint Louis, MO, USA). PAGE Gel Silver Staining Kit (G7210) was purchased from Solarbio (Beijing, China). Cell Counting Kit-8 (CK04) was from Dojindo (Kumamoto, Japan), and CytoTox 96 Non-radioactive Cytotoxicity Assay (G1782) was from Promega (Madison, WI, USA). In Situ Cell Death Detection Kit-TMR red was from Roche Applied Science (12156792910, Mannheim, Germany). Protein ladder was purchased from Thermo (26616), (Vilnius, Lithuania). DAB substrate kit was from ZSBIO (ZLI-9017, Beijing, China), and hematoxylin was from Solarbio (G4070). All the antibodies against CD3E (512415), CD8A (510793), Granzyme B (252579), and Perforin (500093) were purchased from ZEN BIO (Chengdu, China). The A549 (human non-small cell lung cancer cells), Jurkat (human acute T cell leukemia cells), B16-F10 (mouse melanoma cells) and YAC-1 (mouse lymphoma cells) cell lines were from American type culture collection (ATCC).