Endothelial Cell Protein Extraction and Western Blot

Total protein was extracted from endothelial cells using the same procedures as described in detail elsewhere^{41 (link)}. The protein concentrations were determined by BCA Protein Assay kit (Bio-Rad, Mississauga, ON, Canada). Equal amounts of protein lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes followed by block with 5% skimmed milk at room temperature for 2 h. Subsequently, the membranes were incubated with the primary antibodies against NLRP3 (Proteintech, Chicago, USA, 1:1000, Cat. No.: 19771-1-AP), ASC (Santa Cruz, USA, 1:500, Cat. No.: sc-22514-R), Caspase-1 (Proteintech, Chicago, USA, 1:1000, Cat. No.: 22915-1-AP), IL-1β (ABclonal, Boston, USA, 1:1000, Cat. No.: A1112), IL-18 (ABclonal, Boston, USA, 1:1000, Cat. No.: A1115), or GAPDH (Proteintech, Chicago, USA, 1:2000, Cat. No: 60004-1-lg) at 4 °C overnight. After washing with PBST three times, the membranes were incubated with the fluorescence-conjugated anti-rabbit IgG secondary antibody (1:10,000) for 1 h. Western blot bands were examined and analyzed by Odyssey Imaging System (LI-COR, Inc., Lincoln, NE, USA).