Anti nlrx1

The antibodies used in this study were as follows: anti-GST (Santacuze, #SC-138), anti-V5 (Invitrogen, #46–0705), or anti-IRF3 (Abcam, #ab25950), anti-phospho-IRF3 (Ser 396) (Cell signaling, #4947), anti-NF-κB p65 (Cell signaling, #4764), anti-phospho-NF-κB p65 (Ser536) (Cell signaling, #3031), anti-STAT1 (Cell signaling, #9175), anti-phospho-STAT1 (Cell signaling, #9167), anti-phospho-p38 (Cell signaling #9216), phospho-TBK1 (Cell signaling #5483), anti-NLRX1 (Proteintech, #17215-1-AP) and anti-His (Santacuze, #SC-1803) antibodies. The anti-FAF1 monoclonal antibody was provided by Dr. Eun-hee Kim (Department of Biology, Chungnam National University, Korea). The anti-interferon-α/β receptor (IFNAR) (25 μg/ml; Leinco Technologies) was pre-incubated in RAW264.7 cells and MEFs for 1 hr before VSV-GFP infection to block IFNAR.

Protein extraction was performed by lysing the cells in Laemmli sample buffer and separated by SDS-PAGE using 7.5% polyacrylamide gels and electrotransferred to nitrocellulose membranes (Bio-Rad, Cat. No. 162-0115). Non-specific binding sites were blocked with 5% non-fat dry milk diluted in TBS Tween buffer (50 mM Tris, 0.5 M NaCl, 0.05% Tween-20, pH 7.4). The following antibodies were used for protein detection: anti-RIG-I (Cell Signaling, Danvers, MA, USA, Cat. No. 3743), anti-MDA5 (Cell Signaling, Cat. No. 5321), anti-TBK1 (Cell Signaling, Cat. No. 3504), anti-MAVS (Cell Signaling, Cat. No. 3993), anti-NLRC5 (clone 3H8, Millipore, Cat. No. MABF260), anti-NLRX1 (Proteintech Group, Manchester, UK, Cat. No. 17215-1-AP), anti-IκBα (Cell Signaling, Cat. No. 4812), and anti-β-actin (Santa Cruz Biotechnology, Cat. No. sc-47778). The bound antibodies were labeled with anti-mouse (Bio-Rad, Cat. No. 1721011), anti-rat (Bio-Rad, Cat. No. 5204-2504) or anti-rabbit (GE Healthcare, Cat. No. NA934) horseradish peroxidase-conjugated secondary antibodies and were visualized by the ECL system using SuperSignal West Pico or Femto chemiluminescent substrates (Thermo Scientific, Rockford, IL, USA, Cat. No. 34580 and 34095) and X-ray film exposure. Densitometric analysis of immunoreactive bands was performed using Image Studio Lite Software version 5.2 (LI-COR Biosciences, Lincoln, Nebraska USA).

Lung tissues were gently homogenized and lysed in RIPA lysis and extraction buffer (Thermo Fisher). The protein concentrations were measured using the Bradford assay. Equal amounts of protein samples were loaded onto 8–12% SDS-PAGE gels and separated by electrophoresis and then transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). Membranes were blocked in Tris buffered saline containing 0.1% Tween 20 (TBST) with 5% skim milk and incubated overnight at 4 °C with primary antibodies. After washing with TBST, the membranes were incubated at room temperature with secondary antibodies for 1 h. The protein signal was analyzed using Image J software. Protein samples were normalized to GAPDH. The primary antibodies were used as follows: anti-NLRX1 (Proteintech, Rosemont, IL), anti-BAX, anti-Cyto C, anti-P-ERK 1/2, anti-T-ERK 1/2, anti-P-JNK, anti-T-JNK, anti-P-p38, anti-T-p38, and anti-GAPDH (Cell Signaling Technology, Danvers, MA).