Anti mettl14

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.

Total protein from tissues or cell lysates was extracted using a buffer containing 1% phenylmethylsulfonyl fluoride (PMSF), and protein concentrations were determined using a bicinchoninic acid (BCA) assay kit (Beyotime Institute of Biotechnology). Protein (40 μg/lane) was denatured and isolated by SDS (10%)‐polyacrylamide (PAL) electrophoresis (140 V, 50 min). The protein was transferred to a polyvinyl formal (PVF) membrane (350 mA, 90 min) blocked with 5% milk (0% fat), which was then sealed in a container at 37 °C for 60 min. The membrane was subsequently incubated with 5% milk (0% fat) containing the following primary antibodies at 4 °C for 12 h: anti‐METTL14 (1:1000, 51104S, Cell Signalling Technology), anti‐runt‐related transcription factor 2 (RUNX2, 1:1000, 12556S, Cell Signalling Technology), anti‐β‐catenin (1:1000, 8480S, Cell Signalling Technology), anti‐phospho‐GSK3β (1:1000, 9322S, Cell Signalling Technology), anti‐GSK3β (1:1000, 9315S, Cell Signalling Technology) and anti‐β‐actin (1:1000, 3700S, Cell Signalling Technology). Membranes were rinsed three times using Tween‐20 buffer for 5 min each and incubated using the corresponding secondary antibodies at 37°C for 1 h. Density was measured using ImageJ software (National Institutes of Health), and protein bands were normalized to β‐actin.

Lung tissue was lysed in a RIPA buffer (Beyotime Biotechnology, China) with PMSF (Thermo Fisher, USA); and a phosphatase inhibitor cocktail (Thermo Fisher, USA) was also added to the RIPA buffer when p-eNOS was to be analyzed. The lysate was then centrifuged for 10 min at 12,000g, and the supernatant was collected. An enhanced BCA protein assay kit (Beyotime Biotechnology, China) was used to measure the protein concentration. A 10% SDS-PAGE gel was used for electrophoresis, and the electrotransfer occurred for 90 min at 110 V with 0.45 µm PVDF membranes (Merck Millipore, USA). The membranes were incubated with antibodies, and a chemiluminescence method was used for color development. The G:BOX system (Syngene, USA) was used to generate gray-scale images. Image-Pro Plus software (Media Cybernetics, USA) was used to quantify the amount of protein as gray value. The primary antibodies used in the western blots were as follows: anti-METTL3 (1:1000, ab195352, Abcam, UK), anti-METTL14 (1:2000, #51104, Cell Signaling Technology, USA), anti-WTAP (1:1000, #56501, Cell Signaling Technology), anti-FTO (1:1000, ab92821,Abcam), anti-ALKBH5 (1:500, 16837–1-AP,Proteintech, USA), anti-VEGF (1:200, 26157–1-AP,Proteintech), anti-eNOS (1:1000, ab199956,Cell Signaling Technology), anti-p-eNOS (1:1000, #9570, Cell Signaling Technology), and anti-β-actin (1:5000, A1978, Sigma-Aldrich, USA).