Ab181090

Protein levels were determined using Western blot. Briefly, cells or tissues were sonicated in RIPA lysis buffer and homogenized. Cell debris was removed, and the supernatant was obtained by centrifugation for 10 min at 12,000 g and 4 °C. After electrophoretic separation and transfer of bands to a polyvinyl difluoride (PVDF) membrane, the proteins on the membrane were probed with antibodies against c-caspase3 (#9661; Cell Signaling Technology), c-caspase8 (#9496; Cell Signaling Technology), c-PRAP (#5625; Cell Signaling Technology), Rac1 (#4651; Cell Signaling Technology), p22^phox (ab80896; Abcam, Shanghai, China), p47^phox (ab181090; Abcam), NOX4 (ab133303; Abcam), and PKC (#2056; Cell Signaling Technology). Glyceraldehyde phosphate dehydrogenase (GAPDH; ab181602; Abcam) was used as loading controls. The total protein level was normalized to the GAPDH protein level.

In order to determine the position of platelet plasma membrane in sucrose fraction, the presence of specific protein markers was evaluated by immunoblot assay in the control group.
Rabbit monoclonal anti-Flotillin-1 (ab411927, Abcam, Paris, France) anti-GpIb (ab192541, Abcam, Paris, France), anti-NCF1 (ab181090, Abcam, Paris, France) and rabbit polyclonal anti-COX IV (ab16056, Abcam, Paris, France) were used as markers of cholesterol rich microdomains, platelet plasma membrane, cytosol and mitochondria, respectively.
Protein levels in each fraction were quantified by BCA Protein Assay (Thermo Scientific France, Villebon sur Yvette, France). Three microliters of each fraction, containing the same quantity of proteins, were put on nitrocellulose membrane directly. After blocking non-specific binding sites for 1 h with BSA 5% in Tris Buffered Saline (TBS)-Tween (TBS with 0.1% Tween 20), membranes were incubated by primary antibodies, and horseradish peroxidase conjugated goat anti rabbit IgG Heavy & Light (ab6721, Abcam, Paris, France) was used as secondary antibodies.

Protein was extracted from the tissue homogenate and macrophages using RIPA lysis buffer (Sigma-Aldrich). NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) were used to prepare the cytosolic fraction and nuclear extracts. Equivalent quantities (25 µg) of protein were separated by SDS-PAGE gel, transferred onto a nitrocellulose membrane (Millipore), blocked with 5% skim milk overnight at 4 °C, followed by overnight incubation with primary antibodies against Atox1 (1:10000; Abcam, ab154179), p47phox (1:1000; Abcam, ab181090), NLRP3 (1:500; Abcam, ab263889), Caspase 1 p20 (1:1000; Invitrogen, PA5-99390), iNOS (1:20000; Abcam, ab178945), IL-12p40 (1:1000; Abcam, ab133752), IL-10 (1:2000; Abcam, ab1333575), Arg-1 (1:1000; Abcam, ab2333548), Lamin B1 (1:1000; Abcam, ab229025), and β-actin (1:5000; Proteintech Group, Inc., 60066-1-AP) at 4 °C. Subsequently, the membranes were washed thrice with Tris-buffered saline with 0.1% Tween-20 and incubated with the HRP-conjugated secondary antibody (1:10000; ZSGB-BIO, ZB-2301, ZB-2305) for 1 h at 37 °C. Signals were visualized with an enhanced chemiluminescence system.