Bca assay

Total proteins were extracted from synovial tissues and cells by RIPA lysis buffer (Beyotime Institute of Biotechnology), the extracted protein levels were determined by BCA assay (Yeasen Biotech, Shanghai, China). Equal amounts of total protein were separated by SDS-PAGE, followed by transfer to the PVDF membranes. After blocked by 5% skim milk, the membranes were incubated with primary antibodies at 4 ◦C overnight. And incubated with the peroxidase-conjugated secondary antibody for 1 h the next day. All membranes were imaged with ECL super (Sparkjade, Shandong, China). The following antibodies were used: GPR65 (Cat#ER1910-13, Huabio, Hangzhou, China); GAPDH (Cat#AP0063, Bioworld, Nanjing, China); E-cadherin (Cat#R22490, Zen Bioscience, Chengdu, China); N-Cadherin (Cat# R23341, Zen Bioscience, Chengdu, China); Vimentin (Cat#R22775, Zen Bioscience, Chengdu, China).

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.

Lung tissues were weighted and lysed in ice-cold RIPA buffer (P0013B, Beyotime) supplemented with phosphatase inhibitor complex (C500017-0001, Sangon) and protease inhibitor cocktail (C600387-0001, Sangon) according to the manufacturer’s instruction. After homogenization, samples were slowly rotated at 4 °C for 15 min for complete lysis. Then samples were centrifuged and the supernatants were collected. Protein concentrations were determined using the BCA assay (20201ES86, Yeasen). Proteins were separated using SDS polyacrylamide gels and transferred onto nitrocellulose membranes (10600001, Amersham Protran). The membranes were blocked with 5% BSA (A600332, Sangon) in TBS-T buffer (containing 0.1% Tween-20) for 1 h. Then incubated with primary antibodies at 4 °C overnight, and secondary antibody at room temperature for 1 h. The following primary antibodies were used in this study: Rabbit anti-β-ACTIN antibody (1:1000, 4967, Cell Signaling Technology), Rabbit anti-GUCY1A1 antibody (1:500, G4280, Sigma), Rabbit anti-NOTCH3 antibody (1:500, ab23426, Abcam), Rabbit anti-GUCY1B1 antibody (1:1000, ab154841, Abcam). HRP-conjugated donkey anti-rabbit IgG (H + L) (1:10000, 711-066-152, Jackson ImmunoResearch) was used as secondary antibodies. Western blot was performed using Pierce™ ECL Western blotting substrate (32209, Thermo) according to the manufacturer’s instruction.