Anti rig 1

Antibodies used in this study were summarized at the dilutions listed: anti‐METTL14, 1:1000, (IB; Cell Signaling Technology, #: 51104S), anti‐N⁶‐methyladenosine modifications of RNA and DNA (m⁶A), 1:2000, (dot blot; Synaptic Systems, #: 202 003); anti‐IRF3, 1:1000, (IB; Cell Signaling Technology, #: 4302S); anti‐pIRF3, 1:1000, (IB; Cell Signaling Technology, #: 4947S) anti‐TBK1, 1:1000, (IB; Cell Signaling Technology, #: 3504S); anti‐pTBK1, 1:1000, (IB; Cell Signaling Technology, #: 5483S); anti‐RIG‐I, 1:1000, (IB; Cell Signaling Technology, #: D14G6); anti‐MDA5, 1:1000, (IB; Cell Signaling Technology, #: D74E4); anti‐FTO, 1:1000 (IB; Cell Signaling Technology, #: D6Z8W); anti‐METTL3, 1:1000 (IB; Cell Signaling Technology, #: E3F2A); anti‐ALKBH5, 1:1000 (IB; Abcam, #: ab195377); anti‐MAVS, 1:500, (IB; Santa Cruz Biotechnology, #: sc‐365333); anti‐FLAG (M2), 1:1000, (IB; Sigma‐Aldrich, #: F1804); anti‐β‐actin, 1:2000, (IB; ZSGB‐BIO, #:TA‐09). CHX (HY‐12320), actinomycin‐D, (HY‐17559), were obtained from MedChemExpress (MCE, NJ, USA); PMA (P1585) and Dynabeads mRNA Purification Kit (#: 61006) were purchased from Invitrogen; Click‐iT nascent RNA capture kit (#: C10365) was purchased from Life Technologies; EpiMark N6‐Methyladenosine Enrichment Kit (E1610S) was purchased from New England Biolabs.

Protein lysates were extracted from A549 cells treated with 1 μg/mL SLR14 or 100 HA/mL SeV for 1h to 24h, the expression of both RIG-I and GAPDH was measured via western blot, and the difference in RIG-I expression was calculated relative to mock-infected wells. Cells lysed at indicated times with NP-40 buffer containing protease and phosphatase inhibitors (Roche). Equal quantities of lysate were separated on 4-20% SDS-PAGE (Bio-Rad) and transferred onto 0.45 μm^{2 (link)} PVDF membrane, then blocked with TBST + 5% milk before treatment with anti-RIG-I (Cell Signaling Technology) and anti-GAPDH antibodies overnight at 4C. Following washing, blots were treated with HRP-conjugated secondary antibodies and visualized using a blot imager (Bio-Rad). Blots were background-subtracted and the total band intensity was measured via ImageJ, then the ratio of RIG-I/GAPDH expression calculated from both bands.

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).

Recombinant human epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin (IL)-4 were obtained from PeproTech Inc. (Rocky Hill, NJ, USA). Anti-human and anti-mouse monoclonal antibodies for cluster of differentiation (CD)11c, HLA-DR, major histocompatibility complex class II (MHCII), and CD86 labeled with fluorescein isothiocyanate (FITC) or R-phycoerythrin (PE) were purchased from eBioscience (San Diego, CA, USA). Anti-phospho (p)-Smad2 (S465), anti-Smad2, anti-p-Akt (T308), anti-Akt, and anti-RIG-I antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA).