Mettl14

After being washed using PBS, the cells were lysed with RIPA (radioimmunoprecipitation assay) solution containing a protease inhibitor. The acquired proteins were then quantified using a bicinchoninic acid protein assay (BCA) kit, after which 20 mg of total protein was separated on 8% or 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) under electric field. The separated proteins were blotted onto a polyvinylidene difluoride (PVDF) membrane (Millipore, United States), which was then blocked with 5% bovine serum albumin (BSA) solution for 1 h and incubated with primary antibodies against GAPDH (1:1,000, Proteintech, United States), RBM15 (1:1,000, Proteintech, United States), METTL14 (1:1,000, Proteintech, United States), PDCD1 (1:2,000, Proteintech Group, United States), CTLA4 (1:1,000, novus biotechnologies, United States), HNRNRC (1:1,000, abcam, United States), and BTLA (1:1,000, abcam, United States) overnight at 4°C. On the second day, the membrane was incubated with secondary antibodies (1:2,500) at room temperature for 1 h after three times of washes with TBST (Tris-buffered saline-Tween). After being washed for another three times, bands of conjugate proteins were visualized via a ChemiDoc™ MP Imaging system (Bio-Rad Laboratories) with GADPH as the internal control.

The procedures of western blot assay have been described previously.²⁷ Antibodies that recognize AMD1 (11052‐1‐AP), Tubulin (11224‐1‐AP), NANOG (14295‐1‐AP), SOX2 (11064‐1‐AP), KLF4 (11880‐1‐AP), OCT4 (11263‐1‐AP), METTL3 (15073‐1‐AP), METTL14 (26158‐1‐AP), FTO (27226‐1‐AP), and ALKBH5 (16837‐1‐AP) were purchased from Proteintech (Wuhan, China). Antibodies that recognize IQGAP1 (ab86064), phosphoserine (ab9332), phosphothreonine (ab9338), and SPM (ab7318) were obtained from Abcam (Cambridge, MA, USA). Secondary antibodies were obtained from Beyotime Biotechnology (Shanghai, China).

RIP was performed as previously described [33 ]. Briefly, 10⁷ LcLs or Akata cells were collected and lysed with RIP buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 5mM EDTA, 0.5mM DTT, 0.5% NP40, 100 U/ml RNAase inhibitor SUPERase•in, 1×protease inhibitor (539131, Millipore, Burlington, MA). 3μg of METTL14 (Proteintech Group Inc, Rosemont, IL) or IgG (NI01, Millipore, Burlington, MA) was conjugated to protein A/G agarose beads (Thermo Fisher Scientific, Waltham, MA) by incubation for overnight at 4°C in RIP buffer at 4°C overnight. Beads were washed with RIP buffer for three times, followed by DNA digestion at 37°C for 30 min and incubation with 50μg of proteinase K (Thermo Fisher Scientific, Waltham, MA) at 55°C for 30min. Input and co-immunoprecipitated RNAs were recovered by TRIzol, extraction and analyzed by qPCR.

Cells were washed twice with cold PBS. Then the cells were lysed with RIPA lysis buffer (50 × 10⁻³m Tris‐HCl (pH 7.4), 150 × 10⁻³m NaCl, 1% Triton X‐100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, leupeptin) for 30 min. The cell lysate was centrifuged at 12 000 rpm for 15 min. The protein concentration was measured using BCA protein assay kit. Equal amounts of total protein were separated on 10% or 15% SDS‐PAGE and transferred onto NC membranes, blocked with 5% nonfat milk at room temperature for 1 h, and incubated with primary antibodies at 4 ˚C overnight. After washed three times, the membranes were incubated with secondary Ig conjugated HRP for 1 h at room temperature. Protein bands were visualized with the ECL enhanced chemiluminescence regent Kit (NCM Biotech). The following antibodies were used: RBMS1 (Abcam ab150353), S100P (Proteintech 11803‐1‐AP), YTHDF1 (Proteintech 17479‐1‐AP), YTHDF2 (Proteintech 24744‐1‐AP), YTHDF3 (Proteintech 25537‐1‐AP), METTL3 (Proteintech 15073‐1‐AP), METTL14 (Proteintech 26158‐1‐AP), FTO (Proteintech 27226‐1‐AP), m6A (Proteintech 68055‐1‐lg), FLAG (Sigma 1804), V5 (Proteintech 14440‐1‐AP), VINCULIN (Proteintech 66305‐1‐Ig), GAPDH (Proteintech 60004‐1‐Ig).

Total protein was extracted using RIPA buffer (Beyotime, China) supplemented with a protease inhibitor cocktail (Yeason, China). The protein concentration was determined using the bicinchoninic acid (BCA) assay (Yeason, China). The protein samples were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were then incubated with primary antibodies against METTL3, METTL14, ALKBH5, FTO, MIB1 (all from Proteintech, Wuhan, China) at a dilution of 1:1000. The antibodies against HSV viral proteins ICP0, ICP8, and gC were obtained from Dr. Bernard Roizman’s laboratory at The University of Chicago and were used at a dilution of 1:1000. Subsequently, the membranes were incubated with a peroxidase (HRP)-conjugated secondary antibody (1:1000, Cell Signaling Technology, USA). After thorough washing, the protein signals were detected using a chemiluminescence system (Tanon, China) and analyzed using Image Lab Software. The detailed information on the antibodies and related agents used in this study is provided in the Supplementary Table 2.