Bl521a

Western blot was used to analyze the METTL3, NOTCH1, α-SMA, SM22α, and calponin protein expressions after 48 h of transfection. The total protein was extracted from HASMCs using RIPA lysis buffer. Proteins were quantified for each group according to with a bicinchoninic acid (BCA) protein determination kit (BL521A; BioSharp). SDS-PAGE loading buffer was mixed with the mixture, and the mixture was heated in a boiling water bath at 100 ℃ for 5 min. The protein was adsorbed on a polyvinylidene fluoride (PVDF) membrane by gel electrophoresis and sealed with a 5% skim milk solution for 90 min at room temperature. The anti-METTL3 antibody (EPR18810; dilution: 1:1,000; ab195352; Abcam, Cambridge, UK), anti-α-SMA antibody (dilution: 1:10,000; ab5694; Abcam), anti-TAGLN/transgelin antibody (dilution: 1:5,000; ab14106; Abcam), anti-calponin 1 antibody (EP798Y; dilution: 1:5,000; ab46794; Abcam), anti-Notch1 antibody (EP1238Y; dilution: 1:2,000; ab52627; Abcam), and GAPDH (dilution: 1:5,000; ab9485; Abcam) were incubated overnight. We then incubated the horseradish peroxidase (HRP)-labeled secondary antibody immunoglobin G (IgG)-HRP (dilution: 1:2,000; BL003A; BioSharp). Exposure was performed using an ultra-sensitive electrochemiluminescence (ECL) substrate (BL520A; BioSharp). GAPDH was used as an internal reference.

Synovial samples were lysed in RIPA buffer (C5029, Bioss, Beijing, China) containing 1% protease inhibitor PMSF (D10411, Bioss, Beijing, China), the supernatant was centrifuged in centrifuge tubes at 12,000 rpm in a 4 °C centrifuge for 30 min, and the protein concentration was measured using a BCA quantitative kit (BL521A, Biosharp, Hefei, China). After denaturation, the proteins were separated using SDS-PAGE gel (P0015, Beyotime, Shanghai, China) electrophoresis, and the target protein was transferred to a PVDF membrane (0.45μm, GE, USA), with a constant current of 200 mA. The membranes were incubated with IL7R antibody ABP53336 (1:1,000, Abbkine, Wuhan, China) and GAPDH antibody A19056 (1:1,000, ABclonal, Woburn, MA, USA) on a shaking ice bed at 4 °C for 14 h overnight to obtain the primary antibody. The next day, the primary antibody was recovered and incubated with the secondary antibody. Luminescence was performed on a light-emitting machine to capture strip images, and ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used for densitometric analysis of western blots.

Total protein was extracted using a protein extraction kit (#P0013C, RIPA Lysis Buffer, Beyotime). The protein concentration was measured using the BCA method (Biosharp BL521A). Equal amounts of proteins (30 μg) were separated and transferred to PVDF membranes. Following encapsulation in 5 % skim milk, the PVDF membranes were incubated with the following primary antibodies: anti-IRF1 (1:1000, #a7692, ABclonal, Wuhan, China), anti-phospho-(Ser/Thr) Phe (1:1000, Cell Signaling Technology, #9631), anti-STAT3 (1:1000, #10253-2-ap, Proteintech, Wuhan, China), anti-phospho-STAT3-Y705 (1:200, #AP0070, ABclonal), anti-PD-L1 (1:1000, #A1645, ABclonal), and GAPDH (1:50000, #60004-1-Ig, Proteintech). Following incubation with secondary antibodies (#BL003A/#BL001A, Biosharp, Beijing, China), an enhanced chemiluminescence (ECL) system was used to visualize the protein bands, followed by quantification with ImageJ software.