Yt2322

TMJ samples were collected, using 4% paraformaldehyde and 10% EDTA for fixation and decalcification, and following embedded in paraffin, continuous mid‐sagittal sections of 4 μm were cut for subsequent staining. After dewaxing and gradient hydration, haematoxylin and eosin (HE) staining was performed to quantify the synovial inflammation.
Immunofluorescence staining was incubated with the designated primary antibody overnight at 4°C and then conjugated the primary antibody with a fluorescent secondary antibody to visualize staining, and 4′,6‐diamidino‐2‐phenylindole (DAPI) reagent was used to develop nuclei. Primary antibodies were as follows: rabbit anti‐ALPK1 (1:300, 19107‐1‐AP; Proteintech), mouse anti‐CD68 (1:500, 66,231‐2‐Ig, Proteintech), rabbit anti‐Vimentin (1:200, 10366‐1‐AP, Proteintech), rabbit anti F4/80 (1:200, GB11027, Servicebio) and rabbit anti‐INOS (1:500, GB11119, Servicebio).
Immunohistochemical staining was to incubate with the designated primary antibody at 4°C overnight, then with the designated secondary antibody for 30 min, and finally developed the colour with DAB (DAB‐0031, Bio technologies), followed by counterstaining of nuclei with haematoxylin. Primary antibodies were as follows: rabbit anti‐TNF‐α (1:400, 11948S, Cell Signaling Technology), rabbit anti‐IL‐1β (1:400, YT2322, Immunoway) and rabbit anti‐IL‐6 (1:400, A14687, ABclonal).

Cells were first lysed with RIPA lysis buffer to collect cellular proteins. Then, the protein was separated by 10% SDS‐PAGE and then electro‐transferred to PVDF membrane. The protein on the membrane was detected with a suitable primary antibody. Finally, the protein was incubated with the secondary antibody for 1 h, and the protein signal was detected by the luminometer ECL. Primary antibodies were as follows: rabbit anti‐INOS (1:1000, GTX130246, GeneTex), mouse anti‐β‐actin (1:5000, RAB0100, Frdbio), rabbit anti‐IL‐1β (1:1000, YT2322, Immunoway), mouse anti‐PKM2 (1:1000, 60268‐1‐Ig, Proteintech), Mouse anti‐Histone H3.1(1:1000, GB11102, Servicebio), rabbit anti‐TNF‐α (1:1500, GTX110520, GeneTex), rabbit anti‐ALPK1 (1:6000, Abcam) and mouse anti‐GAPDH (1:5000, RAB0101, Frdbio).