Anti his tag

The antibiotics (kanamycin, rifampicin, cefotaxime, and chloramphenicol) and also the Ni-Sepharose™ 6 Fast Flow resin were purchased from the Sigma-Aldrich Co. (Missouri, United States). The Taq DNA Polymerase Master Mix for the PCR experiments was obtained from the Ampliqon Co. (Odense, Denmark). The reagents and enzymes used for RNA extraction, cDNA synthesis, and qPCR experiments were purchased from Thermo Fisher Scientific Co. (Waltham, MA, USA). The primary antibodies (i.e., anti-His-tag, anti-hIL-2, and anti-CmR) and also the goat anti-rabbit IgG (alkaline phosphatase conjugate) antibodies, for western blotting, were respectively purchased from the Santa Cruz Biotechnology Co. (Heidelberg, Germany) and Abcam Co. (Cambridge, MA, USA). The reagents for the ELISA assay were obtained from the Bio-Techne Co. (Minnesota, USA).

For CSNPs-binding, hACE2-293T cells were incubated with 10 µM peptide for 1 h and then treated and incubated with 5 µM SARS-CoV-1 S1 protein-His Tag (AcroBiosystems, S1N-C52H3-100UG, USA) for 24 h. The hACE2 over-expression was evaluated in hACE2-293T cells through mRNA expression level using the following primers: (Cosmo Genentech, hACE2-F: 5′-TCC ATT GGT CTT CTG TCA CCC G-3′, hACE2-R: 5′-AGA CCA TCC ACC TCC ACT TCT C-3′, Republic of Korea). After three times whshing with PBS, the cells were incubated with primary antibody anti-ACE2 (1:100, Cell signaling, 4355S, USA), anti-CTNNB1 (1:100, Cell signaling, 8480S, USA), and anti-His-Tag (1:100, Santa Cruz, sc-8036, USA) at 4 °C for 2 h in serum-free media. The cells were fixed with 4% paraformaldehyde for 5 min and incubated with donkey anti-mouse IgG conjugated with Alexa Fluor 594 (1:200, Thermo, A21203, USA) and donkey anti-rabbit IgG Alexa Fluor 488 (1:200, Thermo, A21206, USA) at room temperature for 2 h. Before the secondary antibodies were incubated, cells were blocked with a blocking solution (1% PBS containing 1% BSA and 0.1% Tween 20) at room temperature for 1 h. All secondary antibodies were diluted in an appropriate concentration of blocking solution. Nuclei were stained with DAPI-containing mounting solution (Vector, H-1200, USA). The cells were then visualized on an LSM710 (Carl Zeiss) confocal microscope.