Anti phospho irf7

WT and Mal^−/− iBMDMs were seeded (1 × 10⁶ cells/mL; 2mL) on a 6-well plate and grown for 24 h. Cells were then treated with 100 nM R848 or 100 ng/mL LPS for designated times. Cells were washed in ice-cold PBS and lysed in HS buffer. Cell lysates were subjected to SDS-PAGE followed by Western blot analysis with an anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-p38, anti-p38, anti-phospho-JNK, anti-phospho-IRF3, anti-IRF3, anti-phospho-IRF7 (Cell Signaling, Danvers, MA, USA), anti-β-actin (Sigma, St. Louis, MO, USA), and anti-IκBα (Santa Cruz Biotechnology, Dallas, TX, USA) antibody, secondary antibodies: IRDye 800CW Goat anti-Rabbit IgG (H + L), IRDye 680RD Donkey anti-Mouse IgG (H + L) (LI-COR, Lincoln, NE, USA). Imaging was performed using ODYSSEY CLx Infrared Imaging System (LI-COR).

The cells were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate and 0.1% SDS) supplemented with protease and phosphatase inhibitors for 30 min on ice. The cell lysates were cleared by centrifugation. The proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane (Whatman/GE). The membranes were probed with antibodies. The following were used for western blotting: antiphospho-IRF3 (Cell Signaling), anti-IRF3 (Cell Signaling), antiphospho-IRF7 (Cell Signaling), anti-IRF7 (Cell Signaling), antiphospho-TBK1 (Cell Signaling), anti-TBK1 (Santa Cruz), antiphospho-IkBa (Cell Signaling), anti-IkBa (Cell Signaling), antiphospho-p38 (Cell Signaling), anti-p38 (Cell Signaling), antiphospho-JNK (Santa Cruz), anti-JNK (Santa Cruz), antiphospho-ERK (Santa Cruz), and anti-actin (Santa Cruz). Visualization was performed on film using ECL Plus (GE Healthcare).

The isolated OCs were fixed in 4% paraformaldehyde (Sigma–Aldrich, Cat# 158127) in PBS (Sigma–Aldrich, Cat# P4417), permeabilized by washing with 0.1% Triton X-100 (Sigma–Aldrich, Cat#X100) in PBS, and then incubated 1 h at room temperature with rabbit monoclonal anti-phospho-Irf7 (Cell Signaling, Cat#24129). Following a 1-h incubation, samples were washed and goat anti-rabbit Alexa Fluor 647 sary antibody (Thermo Fisher Scientific, Waltham, MA, USA, Cat#A21245) was added for 1 h at room temperature, plus 1:100 diluted solution of Alexa Fluor 488–labeled phalloidin (Molecular Probes, Cat#A12379) in PBS for 40 min at room temperature. Samples were washed with PBS and incubated with DAPI for 5 min. The OCs were then washed with PBS and mounted on microscope slides with Mowiol (Sigma–Aldrich, Cat#475904) for visualization and image capture.