Ab50772

Eight-well Lab-Tek II chamber slides (Nunc) were seeded with RIP1 knockdown stable clones of A549 cells (4 × 10⁴/well), and the cells were transduced with vAcE baculovirus at an MOI of 50 for various times as indicated. 24 hpt, the cells were washed three times with Dulbecco’s PBS (DPBS; Invitrogen) and fixed with 4% paraformaldehyde for 10 min. Cells were washed three times with DPBS buffer and then permeabilized by incubation with −20°C 100% acetone for 10 min. After three 5-min washes in DPBS, the cells on the slides were blocked with blocking buffer (10% FBS in DPBS) for 1 hr and then incubated with the appropriate primary antibodies (1:150 dilution in blocking buffer) overnight at 4°C. Primary antibodies were pNF-κB (Ser⁵³⁶) (3033, Cell Signaling Technology), IRF3 (ab50772, Abcam), or IRF7 (ab62505, Abcam). Cells were then washed three times with washing buffer (0.1% Tween 20 in DPBS, DPBS-T) and incubated for 1 hr in the dark with 1:200 dilutions of Alexa Fluor 405 goat anti-mouse immunoglobulin G (IgG) for IRF3 or Alexa Fluor 555 goat anti-rabbit IgG for pNF-κB and IRF3 (Invitrogen). Cells were then stained with Hoechst 33342 (Invitrogen) for 15 min before being washed three times in washing buffer and sealed with aqueous mounting medium (HIS002B, Serotec). Fluorescent images were visualized with a Zeiss laser confocal microscope (LSM510).