Gtx125989

Antibodies used in the study included the following: rabbit anti-YTHDC1 (GTX32976, GeneTex, USA); rabbit anti-YTHDF2 (24744-1-AP, proteintech); rabbit anti-METTL3 (15073-1-AP, proteintech); mouse anti-Flag-tag (F1804, SigmaAldrich, Saint Louis, MO, USA); mouse anti-HA-tag (M180-3, MBL, Japan); mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CB100127, California Bioscience, Coachella, CA, USA); rabbit anti-IAV NP, NS1, and NEP, (GTX125989, GTX125990, and GTX125953, GeneTex, USA); mouse anti-NP (produced in our laboratory); control rabbit IgG polyclonal (AC005, ABclonal Biotechnology, Cambridge, MA, USA); horseradish peroxidase-conjugated anti-mouse and anti-rabbit (BF03001 and BF03008, Beijing Biodragon Inmmunotechnologies, China); Alexa Fluor 594-conjugated goat anti-rabbit (GR200G-43C, Sungene Biotech); fluorescein isothiocyanate (FITC) goat anti-mouse (GM200G-02C, Sungene Biotech); and FITC-goat anti-rabbit (GR200G-02C, Sungene Biotech). 4′,6′-diamidino-2-phenylindole (1:1000) (no. C1002) was purchased from the Beyotime, China.

IAV proteins were detected using polyclonal anti-IAV PB1 protein antibody (GTX125923, GeneTex) and anti-IAV NP protein antibody (GTX125989, GeneTex), diluted 1:2000 in phosphate buffered saline (PBS)/5% BSA (RPI). To confirm fractionation of the A549 cells, Mitotracker (AB92824, Abcam), γ-tubulin (MCA77G, Bio-rad) and Histone H3 (AB1791, Abcam) antibodies were used, diluted 1:1000–5000 in PBS/5% BSA (RPI). Secondary antibodies IRDye 800 goat anti-rabbit (926-32211, LI-COR), and IRDye 680 goat anti-rat (926-68076, LI-COR) diluted 1:10,000 in PBS/5% BSA were used. Western blots were developed using the Odyssey CLx imaging system (LI-COR).

IAV proteins were detected using rabbit polyclonal antibodies anti-PB1 (GTX125923, GeneTex), and anti-NP (GTX125989, GeneTex) diluted 1:2000 in blocking buffer (PBS, 5 per cent bovine serum albumin Research Products International (RPI), 0.1 per cent Tween 20 (RPI)). Cellular proteins were detected using the rat monoclonal antibody anti-tubulin (MCA77G, Bio-Rad) diluted 1:3000 in blocking buffer. Secondary antibodies IRDye 800 donkey anti-rabbit (926-32213, LI-COR) and IRDye 680 goat anti-rat (926-68076, LI-COR) were used to detect Western signals with a LI-COR Odyssey scanner.

Protein samples were collected via chemical cell lysis using RIPA buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, 140 mM NaCl, 0.1% SDS) and normalized by total protein concentration before adding SDS-PAGE sample buffer (Bio-Rad). Protein samples were loaded and run on a 4–20% polyacrylamide gels (Bio-Rad). Gels were transferred to nitrocellulose membranes before being blocked with PBS containing 5% (w/v) non-fat dried milk and 0.1% Tween-20 for at least 1 hour at room temperature or overnight at 4°C. Membranes were incubated with primary antibody diluted in PBS containing 5% (w/v) non-fat dried milk and 0.1% Tween-20 for at least 1 hour at room temperature or overnight at 4°C overnight. Primary antibodies used included anti-N1 (4A5, gift from Gene Tan at J. Craig Venter Institute), anti-NP (GeneTex GTX125989), anti-PB1 (GeneTex GTX125923), and anti-GAPDH (Abcam ab181603). Membranes were washed 3 times with PBS containing 0.1% Tween-20 before being incubated with anti-mouse-HRP (Invitrogen A16072) or anti-rabbit-HRP (Invitrogen A16104) secondary antibodies for 1 hour at room temperature. Membranes were washed 3 times with PBS containing 0.1% Tween-20 before treatment with Clarity or Clarity Max ECL (Bio-Rad) and exposure to film for development. Uncropped Western blots are shown in S10 Fig.

IAV proteins were detected using rabbit polyclonal antibodies anti-PB1 (GTX125923, GeneTex), and anti-NP (GTX125989, GeneTex) diluted 1:2000 in blocking buffer (PBS, 5% bovine serum albumin (RPI), 0.1% Tween-20 (RPI)). Cellular proteins were detected using the rat monoclonal antibody anti-tubulin (MCA77G, Bio-Rad) diluted 1:3000 in blocking buffer. Secondary antibodies IRDye 800 donkey anti-rabbit (926-32213, LI-COR) and IRDye 680 goat anti-rat (926-68076, LI-COR) were used to detect Western signals with a LI-COR Odyssey scanner.

Antibodies used for Western blotting, immunoprecipitation, and indirect immunofluorescence were anti-Flag M2 mouse monoclonal antibody (F3165; Sigma, USA); anti-LYAR mouse polyclonal antibody (H00055646-B01P; Abnova, China); anti-NPM1 rabbit polyclonal antibody (AP2834a; ABGENT, USA); anti-HA, -GFP, and -glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibodies (PMK013C, PKM009S, and PMK043F; PMK Bio, China); anti-Histone 3.1 polyclonal rabbit antibody (p30266; Abmart, USA); rabbit polyclonal antibodies against influenza A viral proteins PB1, PB2, PA, NP, and M1 (GTX125923, GTX125926, GTX118991, GTX125989, and GTX125928; GeneTex, USA); and Alexa Fluor 488-conjugated AffiniPure goat anti-rabbit and Alexa Fluor 594-conjugated affinipure goat anti-mouse secondary antibodies (SA00006-2 and SA00006-3; Proteintech, USA). The small-molecule compounds used in this study were CHX (cycloheximide; 100 μg/ml; 66819; Sigma, USA) and DAPI (4′,6′-diamidino-2-phenylindole dihydrochloride; 1:1,000) (C1002; Beyotime, China).