Mouse anti his tag mab

Antibodies were commercially purchased including mouse monoclonal antibody anti-PGK1 (Santa Cruz Biotechnology, Cat. sc-130335; RRID: AB_627677), mouse monoclonal antibody anti-phosphate-tyrosine (anti-pY; Santa Cruz Biotechnology, Cat. sc-7020; RRID: AB_628123), rabbit polyclonal antibodies anti-acetylated lysine (anti-Ac; Cell Signaling Technology, Cat. 9441), rabbit monoclonal antibody anti-β-actin (ABclonal, Cat. AC026), mouse anti-GFP-Tag mAb (ABclonal, Cat. AE012), mouse anti-His-Tag mAb (ABclonal, Cat. AE003), mouse control IgG (ABclonal, Cat. AC011), and alkaline phosphatase-labeled goat anti-rabbit or horse anti-mouse IgG secondary antibodies (ZSGB-BIO, Cat. ZB-2308; ZB-2310). ATG8 and Caspase3 were detected with rabbit polyclonal antibodies against H. armigera ATG8 and Caspase3 that were prepared in our laboratory.

Specific amino acid sequences of CCV ORF39 (457–474 aa, and 1031–1045 aa) and ORF59 (299–316 aa and 75–90 aa) were initially synthesized and selected, then were coupled with KLH carrier protein. New Zealand white rabbits were vaccinated to collect rabbit serum to prepare rabbit anti-ORF39 and 59 polyclonal antibodies (ABclonal, Wuhan, China), as previously reported [16 (link)]. The following antibodies were purchased commercially: HRP Goat Anti-Mouse IgG (H + L) (ABclonal, China), Mouse anti His-Tag mAb (ABclonal, China), Mouse anti GFP-Tag mAb (ABclonal, China), GAPDH Rabbit mAb (ABclonal, China), HRP Goat Anti-Mouse IgG (H + L) (Beyotime, Haimen, China), and HRP Goat Anti-Rabbit IgG (H + L) (Beyotime, China).

HEK293T cells were transfected with a plasmid DNA encoding SARS-CoV-2 spike protein once cells reached approximately 50–70% confluency using polyethylenimine. 24 hours post transfection, cells were rinsed with PBS then detached with EDTA. After cell counting, 5×10⁵ cells were washed with 1 mL PBFA (PBS supplemented with 2% fetal calf serum and 0.1% sodium azide) by resuspension and centrifugation at 800×g for 3 min at 4°C. The pellet was subsequently incubated in 100 μL primary antibody (sybodies at a final concentration of 3 μM or an equal volume of PBS; diluted in PBFA) at 4°C for 30 min. After washing in PBFA, cells were incubated in 100 μL of secondary antibody (1:100, Mouse anti-His-tag mAb, Abclonal, #AE003; then with Goat Anti-Mouse IgG(H+L)-FITC, SouthernBiotech, #1036–02; diluted in PBFA) at 4°C for 30 min and washed twice with 1 mL PBFA. 100,000 live cells in ice-cold PBS were analyzed by fluorescence-activated cell sorting (FACS). As a control group, the same amount of cells were also analyzed by FACS with no antibodies (sybodies and secondary antibodies).