Horseradish peroxidase hrp conjugated anti flag

For Co-IP assays, total protein extracted from FLAG:HDR1 transgenic or none-transgenic and OsK4-RNAi transgenic or none-transgenic was mixed with purified 2mg HIS-HD1 and anti-FLAG antibody. The mixture was incubated overnight with Magna Protein A Magnetic Beads [Millipore, Temecula, California, USA] at 4°C in 1000 μl of binding buffer (20 mM Tris-HCl, pH 7.6, 2.5 mM β-mercaptoethanol, and 0.1 M NaCl). After incubation, the beads were washed five times with washing buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 0.5% Triton X-100). After washing, 40 μl of 1× SDS-PAGE sample buffer was added, the mixture was denatured, and the sample was loaded on a 10% SDS-PAGE gel. Proteins were detected using a horseradish peroxidase (HRP)-conjugated anti-FLAG [Sigma, St. Louis, MO, USA], anti-HD1 or anti-HIS antibody, and then visualized with an ECL reagent (GE Healthcare).

Goat anti-human ACE2 affinity purified IgG (Cat No. AF933) (1:5000) was from R&D Systems; rabbit anti-human NPC1 (Cat No. ab134113) (1:5000) was from Abcam; rabbit anti-SARS-CoV-spike protein (Cat No. NB100-56047) (1:3000) was from Novus Biologicals; rabbit anti-SARS-CoV-2 spike RBD protein (Cat No. 40592-T62) (1:3000), rabbit anti-Zaire EBOV-GP (Cat No. 40442-T48) (1:5000) were from Sino Biological; mouse monoclonal anti-VSV-G (Cat No. Cat#A02180) (1:2000) was from Abbkine; rat monoclonal anti-FLAG (Cat No. F3165) (1:10,000), horseradish Peroxidase (HRP)-conjugated anti-FLAG (Cat No. A8592) (1:10,000), HRP-conjugated anti-HA (Cat No. H6533) (1:10,000), and HRP-conjugated anti-β-Actin (Cat No. A3854) (1:5000)were from Sigma Aldrich; HRP-conjugated AffiniPure donkey anti-goat IgG (H + L) (Cat. No. 705-035-003), HRP-conjugated goat anti-mouse IgG (Cat No. 115-035-003) (1:5000) and anti-rabbit IgG (Cat No. 111-035-003) (1:5000), and APC-conjugated polyclonal anti-goat antibody (Cat No. 705-136-147) (1:500) were from Jackson ImmunoResearch.