Nbp2 12446

Platelets (10⁷) from Sham or CLP rats or stimulated with or without LPS were allowed to adhere to permanox slides (Lab-Tek, Thermo Fisher Scientific, Waltham, MA). Adhered cells were fixed in 10% formalin for 30 minutes, washed 3Xs with PBS for 10 minutes at RT, and permeabilized with ice-cold 100% methanol for 10 minutes. The slides were blocked in PBS with 10% goat and rabbit serum for 1 hr., washed and incubated with rabbit anti-NLRP3 antibody (NBP2-12446, Novus Biologicals, Littleton, CO) for 2 hours at RT. Slides were then washed and incubated with anti-rabbit Alexa Fluor 488 (A11070, Thermo Fisher Scientific) for 1 hour at RT. The slides were washed again and incubated with Alexa Fluor 647-conjugated rabbit anti-ASC antibody (NBP1-78977AF647, Novus Biologicals) for 1 hour at room temperature. Controls were processed identically, except for omission of the primary antibodies. Preparations were mounted in Cytoseal 60 (Thermo Scientific) and analyzed on a Nikon C1 (Nikon) confocal scanning microscope. The images were captured using a Nikon Eclipse 55i microscope equipped with a Nikon DS-Fi1 color camera (Nikon, Melville, NY).

Platelets (10⁷) from Sham or CLP rats or stimulated with or without LPS were allowed to adhere to permanox slides (Lab-Tek, Thermo Fisher Scientific, Waltham, MA, USA). Adhered cells were fixed in 10% formalin for 30 min, washed 3Xs with PBS for 10 min at RT, and permeabilized with ice cold 100% methanol for 10 min. The slides were blocked in PBS with 10% goat and rabbit serum for 1 h, washed and incubated with rabbit anti-NLRP3 antibody (NBP2-12446, Novus Biologicals, Littleton, CO, USA) for 2 h at RT. Slides were then washed and incubated with anti-rabbit Alexa Fluor 488 (A11070, Thermo Fisher Scientific) for 1 h at RT. The slides were washed again and incubated with Alexa Fluor 647-conjugated rabbit anti-ASC antibody (NBP1-78977AF647, Novus Biologicals) for 1 h at room temperature. Controls were processed identically, except for omission of the primary antibodies. Preparations were mounted in Cytoseal 60 (Thermo Fisher Scientific) and analyzed on a Nikon C1 (Nikon Inc., Melville, NY, USA) confocal scanning microscope. The images were captured using a Nikon Eclipse 55i microscope equipped with a Nikon DS-Fi1 color camera (Nikon) and analyzed using NIS-Elements D 3.0 software.

NSCs were digested in cold RIPA lysis buffer according to the instructions. Following lysate centrifugation and BCA assay, 40 μg of protein was loaded by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene membranes. After being blocked with 5% milk without fat in tris-buffered saline with 0.1% Triton X-100 buffer, the membranes were probed with primary antibodies at 4°C overnight. Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1 : 20000, A0208, Beyotime, Shanghai, China) at room temperature for 1 h. Finally, enhanced chemiluminescence reagents (K12045-d20, Advansta, CA, USA) were used to detect the protein signals. The Alpha Ease FC gel analysis software (Alpha Innotech, CA, USA) was used to calculate the optical density of protein bands in each group. Primary antibodies were used as below: rabbit anti-mouse NLRP3 (1 : 1000, NBP2-12446, Novus Biologicals, CO, USA), rabbit anti-mouse p20 fragment of Caspase-1 (Caspase-1 p20, 1 : 2000, 89332, Cell signaling technology, MA, USA), rabbit anti-mouse N-terminal fragment of cleaved GSDMD (GSDMD-N, 1 : 500, ER1901-37, HUABIO, Hangzhou, China), and anti-β actin (1 : 2000, Proteintech, 20536-1-AP, Wuhan, China).

Protein lysates were added to lyse mouse heart tissues and myocardial cells. The supernatant in different groups was gathered after centrifugation (12,000 rpm for 15 min). The proteins were split by SDS–PAGE before they were devolved into PVDF membranes. After the transfer, we blockaded the membrane with 5% skim milk for 1 h at 37 °C. The primary antibodies against NLRP3 (NBP2-12446, 1:1000; Novus Biologicals, Littleton, CO, USA), caspase-1 (sc-56036, 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), and GSDMD (sc-393581, 1:200; Santa Cruz Biotechnology, Dallas, TX, USA) were washed with TBST solution and blocked overnight at 4 °C. The excess antibody was washed thoroughly with TBST solution 3 times for 10 min. The immunoreactive bands of the secondary antibody to goat anti-rabbit (A0208, 1:2000; Beyotime Biotechnology) and goat anti-mouse (A0216, 1:2000; Beyotime Biotechnology) with the corresponding bound secondary antibody were then visualized with ECL chemiluminescence and quantified by image software. GAPDH, a reference quantity, was used to qualify the protein expression.