Phosphatase inhibitors

Whole cell lysates were prepared from HCAEC using RIPA buffer containing Halt protease inhibitors (Pierce, Rockford, IL, USA) and phosphatase inhibitors (Cayman Chemical, Ann Arbor, MI, USA). The concentration of proteins was determined with the Bradford assay and equal amounts of proteins were loaded per gel pocket. α-tubulin was used as a loading control. Whole cell lysates, mixed with loading buffer, were separated on 10% SDS polyacrylamide gels and electroblotted onto nitrocellulose membranes (Whatman). Membranes were blocked for 1 h in 5 % (w/v) non-fat milk in TBS-T. After blocking, the membranes were probed with rabbit anti-phospho NF-κB p65 and rabbit anti-NF-κB p65 (S536 and E498, respectively, both at dilutions of 1:1000 Cell Signaling Technology, Danvers, MA, USA). As secondary antibodies, horseradish peroxidase-conjugated goat anti-rabbit (Cell Signaling Technology, Danvers, MA, USA) were used. Signals were detected using Femto Luminol (ThermoFisher Scientific, Hempstead, UK) with G:Box (Syngene, Cambridge,UK). Densitometry analysis of protein bands was carried out using the Fusion FX software (Vilber Lourmat). For quantification of Western blots, the levels of phosphorylated NF-κB were normalized to the levels of total NF-κB.

Cell lysates were made from HUVECs using RIPA buffer comprising phosphatase inhibitors (Cayman Chemical, Ann Arbor, MI, USA) and Halt protease inhibitors (Pierce, Rockford, IL, USA). Bradford assay was adopted to test the concentration of proteins. The cell lysates were mixed with loading buffer and separated on a 10% SDS polyacrylamide gels. The proteins on gels were then transferred onto PVDF membrane. The membranes were blocked in TBST containing 0.05 g/mL BSA for 1 h. After blocking, the membranes were incubated overnight at 4 °C with primary antibodies (abcam, san francsico, USA), rabbit-derived GAPDH (1:10000, ab181602), E-selectin (1:200, ab18981), VCAM-1 (1:2000, ab134047), ICAM-1 (1:2000, ab53013), caspase-1(1:500, ab138483), NLRP3 (1:500, ab214185), ASC (1:1000, ab155970), PI3K (1:1000, ab191606), p-PI3K (1:1000, ab182651), AKT (1:10000, ab179463), p-AKT (T308, 1:1000, ab38449) and p-AKT (Ser473,1:2000, 4060 T, Cell Signaling Technology, Boston, USA). The membranes were washed in TBST and then incubated at room temperature for 2 h with goat anti-rabbit IgG (1:5000, Beijing ComWin Biotech Co., Ltd., Beijing, China). The membranes were then rinsed three times with TBST. ECL luminescent solution was used before a chemiluminecence imaging analysis system (GE Healthcare, USA) was applied for observation.

Cell lysates were prepared from HUVECs using a RIPA buffer that contained phosphatase inhibitors (Cayman Chemical, Ann Arbor, MI) and Halt protease inhibitors (Pierce, Rockford, IL). The BCA assay (Thermo Fisher Scientific, Waltham, MA) was used to determine the protein concentrations. The cell lysates were mixed with loading buffer and separated on 10% and 12% SDS polyacrylamide gels. The proteins on the gels were transferred to PVDF membranes. The membranes were blocked in TBST containing 5% BSA for 1 h. After blocking, the membranes were incubated overnight at 4 °C with the primary antibodies: anti-GAPDH (#5174S), anti-caspase-1 (#3866T), anti-GSDMD (#97558S), anti-BCL2 (#15071T), anti-BAX (#5023S) (all 1:1000, Cell Signaling Technology, Inc., Danvers, MA), anti-ASC (#sc-514414, Santa Cruz Biotechnology, Santa Cruz, CA), anti-NOX4 (#14347-1-AP, Proteintech Group Inc., Chicago, IL) and anti-NLRP3 (#ab263899, Abcam, Cambridge, UK). The membranes were washed in TBST and then incubated at room temperature for 2 h with goat anti-rabbit IgG (1:4000, Cell Signaling Technology, Inc., Danvers, MA). The membranes were rinsed three times with TBST, scanned, and analysed by ImageJ software (National Institutes of Health, Bethesda, MD).