Hrp conjugated goat anti rabbit igg h l antibody

Western Blot was performed using standard Western blot methods. c-Fos and Bim were detected using an Anti-c-Fos antibody (ab190289, Abcam, Cambridge, UK) and an Anti-Bim antibody (ab32158, Abcam). c-Fos and Bim primary antibodies were followed by a secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) antibody (A0208, Beyotime, Shanghai, China). β-actin was detected using beta Actin Antibody (HRP Conjugated) (AB2001, Abways Technology, Shanghai, China). Western blot data were analyzed using ImageJ software.

Neutrophils were cultured with opti-MEM (Gibco, USA) in 12-well plates (5 ×10⁵ cells/well) for 12 h and then were stimulated with 1μg/mL SEO for 3 h. Next, 2 mM ATP (Sigma-Aldrich, St. Louis, MO) was added to the cells and incubated for an additional 9 h. After stimulation, the culture supernatants and cell lysates were collected as previously described (21 (link)). Protein concentrations were measured using Bradford protein Assay Kit (Beyotime, Beijing, China). Then proteins were subjected to 12% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane by electroblotting. The membranes were blocked with 5% nonfat dry milk. Then, the membranes were immunoblotted with anti-IL-1β Ab (Bioss, Beijing, China), anti-Caspase1-p20 Ab (AdipoGen, USA), anti-GSDMD Ab (Abcam, Cambridge, UK), anti-JNK mAb (Cell signaling Technology, Danvers, USA), anti-phospho-JNK mAb (Cell Signaling Technology, Danvers, USA), streptavidin-horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) antibody, HRP-conjugated goat anti-mouse IgG (H+L) antibody (Beyotime, Beijing, China). β-actin was employed as a loading control for the cell lysates. Finally, the distinct protein bands were detected by ECL detection reagent (Beyotime, Beijing, China).