Complete phostop

Prior to performing inflammation activation experiments, isolated macrophages were pulsed with ultra-pure LPS (100 ng/ml for 30 minutes; Invivogen/Cedarlane). PMA-differentiated or LPS-pulsed mouse peritoneal macrophages were treated with their respective PXR agonists. Following the designated treatment period, culture supernatants were collected, cells were washed with ice-cold phosphate-buffered saline, and cell lysates were isolated following incubating the cells with lysis buffer (150 mM NaCl, 20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, and protease inhibitor cocktail; phosphatase inhibitor cocktail, Complete Minitab; Complete PhoStop, Roche/Sigma-Aldrich Canada). Total protein was quantified using the Precision Red Advanced Protein Assay (Cytoskeleton/Cedarlane, Burlington, Ontario, Canada), and sample protein concentration was equalized. Culture supernatant and cell lysate samples were resolved, transferred to polyvinylidene difluoride membranes (0.2-μm pores; Bio-Rad Laboratories, Mississauga, Ontario, Canada), and blotted with the following antibodies: anti–caspase-1 (sc-622, sc-56036, and sc392736; Santa Cruz Biotechnology, Dallas, TX). Densitometry was performed using ImageJ, and cleaved caspase-1 was expressed as a percentage of pro–caspase-1.

To isolate cell lysates, culture medium was aspirated, and cells were washed with ice‐cold PBS. Cell lysis buffer (150 mM NaCl, 20 mM Tris (pH 7.5), 1mM EDTA, 1mM EGTA, 1% Triton X‐100, protease inhibitor cocktail: Complete Minitab, and phosphatase inhibitor cocktail: Complete PhoStop (Roche, Laval, Canada)) was then added to cells, and they were frozen at −20°C. Total protein was quantified using the Precision Red Protein Assay (Cytoskeleton, Denver, CO), and protein concentration was normalized between samples. Western blots were performed on normalized protein extracts from Caco‐2 intestinal epithelial cells. Membranes were probed with the appropriate primary antibody (anti‐RAC1, BD Bioscience #610651; anti‐β‐actin, Abcam #mAbcam‐8226) and corresponding horseradish peroxidase (HRP)‐conjugated secondary antibody. Membranes were imaged using the MicroChemi Bio‐Imaging system.