Caspase 8

Brain protein lysates were obtained, as described in [62 (link),63 (link)], and 25 μg of protein lysates was then resolved via immunoblotting for the expression of inflammasome signaling proteins, as described in [64 (link)]. Primary antibodies used in this study were against the following proteins: NLRP1 (Novus Biologicals, Centennial, CO, USA), NLRP3 (Novus Biologicals Centennial, CO, USA), AIM2 (eBioscience, San Diego, CA, USA), NLRC4 (Novus Biologicals, Centennial, CO, USA), pyrin (Santa Cruz Biotechnology, Inc. Dallas, TX, USA), caspase-1 (Novus Biologicals, Centennial, CO, USA), ASC (Santa Cruz), IL-1β (Cell Signaling, Technology, Inc. Danvers, MA, USA), caspase-8 (Novus Biologicals, Centennial, CO, USA), caspase-11 (Novus Biologicals, Centennial, CO, USA), CD63 (Novus Biologicals), β-actin (Sigma-Aldrich, St. Louis, MO, USA) and GAPDH (Sigma Aldrich, St. Louis, MO, USA).

Brain and heart protein lysates were obtained as described in (Mejias et al., 2018 (link)), and lysates were then resolved by immunoblotting for the expression of inflammasome signaling proteins as in (Cyr and de Rivero Vaccari, 2023a (link)). Briefly, lysates were resolved in 4–20% Criterion TGX Stain-Free precast gels (Bio-Rad), using antibodies at a dilution of 1: 1,000. Primary antibodies used in this study were against the following proteins: NLRP1 (Novus Biologicals), NLRP3 (Novus Biologicals), AIM2 (eBioscience), Pyrin (Santa Cruz), caspase-1 (Novus Biologicals), ASC (Santa Cruz), IL-1β (Cell Signaling), caspase-8 (Novus Biologicals), CD63 (Novus Biologicals), and β-actin (Sigma Aldric). Quantification of band densities was done using the UN-SCAN-IT gel 6.3 Software (Silk Scientific Corporation). Membranes were imaged using the ChemiDoc Touch Imaging System (BioRad) following chemiluminescence.

Protein assay was performed using the Pierce Rapid Gold BCA Protein Assay kit (Thermo Fisher Scientific) on snap frozen lung lysate or BAL fluid. 25 ug of lysate or equal volumes of BAL fluid (to add 25 ug of the most concentrated sample) were prepared for each of the antibodies to be used in Bolt LDS sample buffer and Bolt sample reducing agent (Thermo Fisher Scientific) and denatured at 70°C for 10 minutes. Samples and SeeBlue Plus2 pre-stained protein ladder were run on Bolt Bis-Tris 4–12% gel (Thermo Fisher Scientific) and transferred to nitrocellulose. Blots were rinsed with water, imaged and de-stained with 1X tris buffered saline with 0.05% Tween 20 (TBS-T). Blots were then blocked with 5% milk and incubated with primary antibody followed by secondary antibody. Immunoblots were exposed to Super Signal West Pico plus or Fempto chemiluminescent substrate (Thermo Fisher Scientific) and exposed to Amersham HyperFilm ECL (Fisher) film for 1 second to 5 minutes and developed using SRX-101A Medical Film Processor (Konica Minolta). The following primary antibodies were used: caspase-4 (Clone: 4B9; Santa Cruz), caspase-8 (90A992; Novus Biologicals); and GSDMDC1 (64-Y; Santa Cruz). Separate gels were run for each of the antibodies using the same sample preparation on the same day with the same loading control.

The immunoreactivity to caspase-3 (1:400; Novus Biologicals, Littleton, CO), caspase-8 (1:1000; Novus Biologicals, Littleton, CO), caspase-9 (1:50; Santa Cruz Biotechnology, Dallas, TX), caspase-12 (1:200; Novus Biologicals, Littleton, CO), myeloperoxidase (MPO) (1:200; Abcam, Cambridge, UK), and nitrotyrosine (1:200; MilliporeSigma, Burlington, MA) was assessed. Infiltrating neutrophils (MPO-labeled) were counted; caspase-3, caspase-8, caspase-9, caspase-12, and nitrotyrosine expression were semi-quantitatively assessed based on staining intensity and the distribution of the labeled target protein. The analysis was performed under a conventional light microscope in a blinded fashion. The intensity score values were given as follows: 0, no positive staining; 1, weak staining; 2, intermediate staining; and 3, extensive staining, and an area score was assigned as follows: 1 = up to 10% positive cells, 2 = 11–50% positive cells, 3 = 51–80% positive cells, and 4 = more than 80% positive cells, using the ImageJ analysis system (NIH, MD, USA). The total score of each field was calculated as intensity score multiplied by area score (0–12). The evaluation was carried out in four random and nonoverlapping fields of the aorta, and the average value was calculated for each animal.