Anti caspase 1 22915 1 ap

For western blot, cells were lysed by cell lysis buffer (P0013; Beyotime) and cell culture supernatant (serum free) was precipitated by methanol and chloroform. The acquired protein samples were boiled for 10 min at 100 °C before gel electrophoresis. A 5200 Multi Chemiluminescence Imaging System (Tanon) was used to detect the protein bands on blots with Super ECL Detection Reagent (36208ES; Yeasen). The primary antibody used in western blot analysis were as follows: Anti-tubulin (11224-1-AP; proteintech); anti-caspase-11 (14340s; CST); anti-Caspase-1 (22915-1-AP; Proteintech); anti-GSDMD (ab209845; Abcam); anti-IL-1β (ab9722; Abcam).

Cells cultured in 12-well plates were lysed using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China), and a bicinchoninic acid kit (Beyotime Biotechnology) was used to standardize the protein content. SDS-PAGE at 10% or 12.5% was used to separate protein samples. Then, 5% BSA was used to block the nitrocellulose membranes for 1.5 h after sample transfer. Next, the following primary antibodies were added: anti-p30 protein (prepared in our laboratory), anti-Actin (AF0003; Beyotime Biotechnology), anti-TLR4 (293,072; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-MyD88 (TA5195S; Abmart, Berkeley Heights, NJ, USA), anti-p-p65 (3033T; Cell Signaling Technology, Boston, MA, USA), anti-p-P38 (4511T; Cell Signaling Technology [CST], Danvers, MA, USA), anti-p-ERK (4370T; CST), anti-NLRP3 (M035175S; Abmart), anti-ASC (bs-6741R; Bioss, Woburn, MA, USA), anti-caspase-1 (22915-1-AP; Proteintech, Rosemont, IL, USA), anti-GSDMD (20770-1-AP; Proteintech), and anti-GSDMD-N (DF12275; Affinity Biosciences, Cincinnati, OH, USA). After washing thrice with TBST, the membranes were incubated with goat anti-mouse or anti-rabbit second antibodies (LI-COR Biosciences, Lincoln, NE, USA), then imaged with the Tanon-5200 multi-infrared imaging system (Shanghai Tianneng Technology Co., Ltd., Shanghai, China).