Gtx55114

The total protein in the cells was extracted using RIPA lysis buffer and quantified using the BCA Protein Quantification Kit (Yeasen Biotechnology Co., Ltd., Shanghai, China). Medium amounts of proteins were prepared, separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to a nitrocellulose membrane. The membranes were blocked in 5% bovine serum albumin solution at room temperature for 1 h and incubated overnight at 4 °C with primary antibodies specific for IL1B (1:1000, NBP1-42767, Novus Biological Inc., Littleton, CO, USA), phospho–NF–κB p65 (Ser536) (1:2000, GTX55114, GeneTex), NF-κB p65 (1:2000, #8242, Cell Signaling Technologies, Beverly, MA, USA), and GAPDH (1:1000, #2118, Cell Signaling Technologies). The membranes were then incubated with HRP-conjugated goat anti-rabbit IgG H&L secondary antibody (1:5000, ab6721, Abcam) for 1 h at room temperature and developed using an ECL reagent (Abcam). ImageJ software was used to quantify protein band densities. The densities of individual protein bands were normalized to those of the corresponding GAPDH band.

The sections were incubated with 3% H₂O₂ for 10 min at room temperature to block endogenous peroxidase activity, followed by heat-mediated antigen retrieval using a sodium citrate buffer. The sections were sealed for 60 min at room temperature using 5% normal goat serum and incubated with diluted primary antibodies against IL-1B (1:200, ab283822, Abcam, Cambridge, UK), Phospho–NF–κB p65 (Ser536) (1:200, GTX55114, GeneTex, Inc., Alton Pkwy Irvine, CA, USA), and CD31 (1:2000, ab182981, Abcam) at 4 °C overnight, followed by incubation with goat anti-rabbit horseradish peroxidase (HRP)-conjugated IgG H&L (1:2000, ab6721, Abcam) for 1 h at room temperature. Color development was performed using diaminobenzidine, and counter-staining was performed using hematoxylin. The positive signal of the staining (tan color) was quantified using ImageJ software.