L selenomethionine

The AB wild-type (WT) adult zebrafish were maintained in a circulation filtration system (28 ± 0.5 °C, pH 7.0–8.0, conductivity of 500–800 µS/cm) with a light:dark ratio of 14:10 h and fed with hatched fairy shrimp three times per day. Embryos were obtained by natural spawning and cultured at 28.5 °C in an incubator. The embryonic and larval ages were expressed as hours post-fertilization (hpf) or days post-fertilization (dpf). The reagents and antibodies used in the study were as follows: L-selenomethionine (Sigma-Aldrich, St. Louis, MO, USA), TUNEL Bright-Red Apoptosis Detection Kit (Vazyme, Nanjing, China), DAPI, FerroOrange (Sigma-Aldrich, St. Louis, MO, USA), NAC, Reduced GSH, Fer-1 and CDDP (MCE, Monmouth Junction, NJ, USA). Antibodies used were phospho-H3 (PH-3) (Cell Signaling Technology, Danvers, MA, USA), Caspase3 and FITC Goat Anti-Rabbit IgG (ABclonal, Wuhan, China).

Selenomethionine (Se-Met) Tt Pah2 was generated by transforming the Tt Pah2/pET28 plasmid into B834 (DE3) cells (Novagen, Cat. No. 69-041-3), which are auxotrophic for methionine. An overnight culture grown in Luria Broth was added to M9 minimal media supplemented with 19 standard amino acids, l-selenomethionine (50 mg/L), and Kao and Michayluk Vitamin Solution 100× (Sigma-Aldrich). Cells were grown to OD₆₀₀ of 0.6 and induced with 100 μM IPTG at 15 °C overnight before harvesting. Se-Met Tt Pah2 was purified as described for the native protein.

The engineered hAPN and pAPN ectodomains contain extracellular residues 36 to 967 and 36 to 963, respectively. They have a hemagglutinin (HA; YPYDVPDYA) peptide at the N terminus. Soluble ectodomains were produced in stably transfected CHO-Lec 3.2.8.1 (CHO-Lec) cells with selective culture media35 (link). A pAPN protein with Met residues replaced by seleno-Met (Se-Met pAPN) was produced using methionine- and glutamine-free DMEM (Invitrogen) supplemented with 10% dialyzed fetal calf serum (FCS) and L-seleno-methionine (both from Sigma). APN proteins secreted to culture supernatants were purified by affinity chromatography with anti-HA 12AC5 mAb (Roche), followed by size exclusion chromatography in HEPES-saline buffer (20 mM HEPES, 150 mM NaCl) pH 7.5.
Preparation of most soluble CoV S proteins used here has been described16 (link)35 (link). S1H and S3H proteins were derived from the HOL87 porcine CoV S glycoprotein and bear the pAPN-binding domain. Soluble TGEV RBD was derived from the S glycoprotein of the TGEV SC11 strain (GenBank acc. n° AJ271965). It contains S residues 505 to 657, an N-terminal HA peptide, and a FLAG sequence (monovalent RBD variant) or human IgG1 Fc (bivalent RBD-Fc variant) at the C-terminal end. CoV S proteins were produced in CHO-Lec or 293 T cells and purified as described35 (link).