Anti dsrna antibody

The protein expression levels of TGEV N protein in candidate gene KO cells and control cells were determined via an immunofluorescence assay. Cells grown on the 12-well cell culture plates were first infected with TGEV at different MOIs. At 24 hpi, cells were fixed with 4% paraformaldehyde at room temperature for 30 min and then permeabilized at room temperature for 10 min with cold 0.3% TritonX-100 in phosphate buffered saline (PBS). Cells were incubated with TGEV N antibody (A rabbit anti-TGEV N protein polyclonal antibody was prepared in our laboratory) or anti-dsRNA antibody (SCICONS, #10010200, 1:1,000) at 4°C overnight, and the primary antibodies were recognized by Alexa Fluor 594 Goat anti-Mouse IgG (H+L) (Invitrogen, #A-11005, 1:1,000), Alexa Fluor 594 Anti-Rabbit IgG (H + L) (Invitrogen, #A-11012, 1:1,000), or Alexa Fluor Plus 647 Goat anti-Mouse IgG (H+L) (Invitrogen, #A32728, 1:1,000). Cell nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) (Sigma, #D9542) at room temperature for 1 min in the dark. Cells were observed and imaged with a fluorescence microscope (Thermo Fisher Scientific EVOS FL Auto). The proportion of positive cells were calculated using Image J software (three independent wells were imaged, and one random field of view per well was captured for each experimental phase).

Binding assays were performed with binding buffer (20 mM Tris–HCl (pH 8.0), 1.5 mM MgCl₂, 70 mM KCl and 1.5 mM DTT) at 37 °C for the indicated time. To isolate RIG-I/poly (I:C) complexes, magnetic bead-antibody-dsRNA combination was designed. Protein G magnetic dynabeads (5 µl, Thermo Fisher) were mixed with 1 µg of anti-dsRNA antibody (SCICONS, K1) at room temperature for 40 min. Beads-antibody complexes were washed three times, then 1 µg of poly (I:C) was added and incubated at 4 °C for 1 h. Magnetic bead-antibody-dsRNA complexes were washed three times. Beads were incubated with 1 µg of recombinant RIG-I (insect cells) or purified RIG-I (293 T cells) at 37 °C to generate RIG-I-poly(I:C) complexes for the indicated times. For binding kinetics, the complexes were subjected to SDS-PAGE following immunoblotting with anti-Flag. The membranes were cut as proper size prior to immunoblotting with the antibody. To examine dissociation kinetics, the complexes formed for 60 min were incubated with or without 1 mM ATP for the indicated times and the complexes remained on the beads were analyzed as described above.

Marc-145 cells were cultured in 12-well plates and treated with tubercidin and PRRSV at the indicated time points. Then, cells were fixed with 4% paraformaldehyde (Biosharp, China) for 10 min and permeabilized with 0.5% Triton X-100 (Solarbio, China) for 15 min at room temperature. After being washed by PBS three times, cell samples were blocked by 3% bovine serum albumin for 30 min at 37°C. Then, cells were incubated with primary antibodies at 4°C overnight. Next, cells were cultured with indicated secondary antibodies for 1 h avoiding exposure to light and incubated with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Beyotime, Shanghai, China) for another 5 min. The fluorescence of the target proteins was observed using a confocal laser scanning microscope (TCS SP8 STED; LEICA, Wetzlar, Germany) or an inverted fluorescence microscope (UHGLGPS; Olympus, Tokyo, Japan). The primary antibodies used in immunofluorescence assay (IFA) included an anti-PRRSV N antibody, an anti-NF-κB p65 antibody, an anti-dsRNA antibody (Scicons, Szirák, Hungary), and an anti-nsp2 antibody.