E7h9g

RAW 264.7 cells were seeded at 3 × 10⁵ cells/cm² into 12-well plates. On the following day, serum-starved cells were primed with EMD for 1 h and then exposed to LPS for another 6 h. Extracts containing SDS buffer with protease and phosphatase inhibitors were separated by SDS-PAGE (cOmplete ULTRA Tablets and PhosSTOP; Roche, Mannheim, Germany) and transferred onto PVDF membranes (Roche Diagnostics, Mannheim, Germany). Membranes were blocked and the binding of the Caspase-1 (D7F10), gasdermin D (E8G3F), and cleaved gasdermin D (E7H9G) first antibodies (rabbit IgG, 1:1000; Cell Signaling Technology, Danvers, MA, USA) were detected with the second antibody labelled with HRP (goat anti-rabbit IgG, 1:10,000; Cell Signaling Technology, Danvers, MA, USA). After exposure to the Clarity Western ECL Substrate (Bio-Rad Laboratories Inc., Hercules, CA, USA) chemiluminescence signals were visualized with the ChemiDoc imaging system (Bio-Rad Laboratories Inc., Hercules, CA, USA). Quantification of band intensity was performed using ImageJ software.