Ha tag antibody

Immunoprecipitation assay (RIPA) lysis buffer (Solarbio) with phenylmethylsulfonyl fluoride (PMSF; Solarbio) was used to lyse cells. Bicinchoninic acid (BCA) protein assay kit (Solarbio) was used to quantify the protein. AminoLink Plus Coupling Resin was incubated with Flag tag antibody (10 μg; proteintech) or HA tag antibody (10 μg; proteintech) for 120 min at room temperature on a rotator. The complex was incubated with the cell lysate for 2 h. After elution, the sample was loaded into sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) for separation. The protein in the gel was transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After being blocked with skimmed milk for 1 h at room temperature, membrane was incubated with primary antibodies overnight at 4°C and then incubated with secondary antibodies for 1 h at 37°C. Enhanced chemiluminescence (ECL) reagent was used to visualize the immunoblot bands, and optical density of bands was measured using Gel‐Pro‐Analyzer software. Primary antibodies included Flag tag antibody (1:5000; proteintech), HA tag antibody (1:5000; proteintech) and Myc tag antibody (1:2000; proteintech), and secondary antibodies included horseradish peroxidase (HRP)‐labelled goat anti‐rabbit immunoglobulin G (IgG) (1:3000; Solarbio) and HRP‐labelled goat anti‐mouse IgG (1:3000; Solarbio).

Dextran sulfate sodium salt (DSS) was purchased from MP Biomedicals (CA, USA), adenosine triphosphate (ATP) was obtained from In vivoGen Biotechnology (CA, USA), Methyl sulfoxide (DMSO), lipopolysaccharide (LPS), and thioglycolate were purchased from Sigma-Aldrich (MO, USA). Carbobenzoxy-Leu-Leu-leucinal (MG132), chloroquine (CQ), and cycloheximide (CHX) were purchased from Selleck (TX, USA). Dithiothreitol (DTT), ethylene diamine tetraacetic acid (EDTA), and primers used in this study were synthesized by Sangon Biotech (Shanghai, China). Antibodies against NLRP3, Caspase-1, and ASC were purchased from Adipogen Life Sciences (CA, USA). Antibody specific to IL-1β was obtained from Cell Signaling Technology. GSDMD antibody was purchased from Abcam (Cambridge, UK). The Flag-tag antibody was obtained from Genscript (Nanjing, China). β-actin and HA-tag antibodies were purchased from Proteintech (Wuhan, China).

Retroviruses pseudotyped with the spike from Ro-BtCoV HKU10, YN7560 and MERS-CoV were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electron microscopy. Pseudovirus containing supernatants were analyzed and transferred to PVDF membrane. HA tag antibodies (Proteintech, Wuhan, China) and P24 antibodies (Keyuananbo, Wuhan, China) were used to detect recombinant S proteins with a C-terminal HA tag and HIV P24 protein, respectively. Pseudovirus containing supernatants were 0.45 μm filtered and centrifuge at 128,000 g for 2 h, and then resuspend the pellet. We loaded 5 μL of purified pseudoviruses into the grids and incubated for 3 min, and 5 μL of 1% phosphotungstic acid was applied for negative staining. Pseudovirus particles were examined in a H-7000FA transmission electron microscope. The production process, measurements of infection and luciferase activity were conducted as described previously [7 (link),37 (link)].