Flag tag (flag)

HEK-293T, Huh7, and HeLa-ACE2 cell lines were cultured using DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin (Gibco). The Calu-3 cell line was cultured using alpha-MEM (Gibco) supplemented with non-essential amino acids, sodium pyruvate, and FBS (10%). The plasmids were adjusted to 3 μg for 6 well plates and 6 μg for 60 mm dishes, then mixed with polyethylenimine (PEI) (Sigma) transfection reagent in a 1μg:3μL ratio of plasmid:transfection reagent following the manufacturer's recommendations. After more than 30–36 h, cells were subsequently washed twice with PBS. Each step was performed by centrifugation at 600 × g, 4  °C for 5 min. Cell pellets were lysed with 2 × SDS sampling buffer and boiled at 95 °C for 15–30 min. Flag, c-myc, GAPDH, tublin, and actin antibodies were purchased from Transgene; EWSR1 antibody was purchased from Abcam; NP antibody was purchased from Sino Biological Inc; strep-tag II antibody was purchased from GenScript.