Ab38449

Tissues and cells of human were lysed in radioimmunoprecipitation assay buffer (Beyotime) containing a protease and phosphatase inhibitor cocktail (K1015, APExBIO). The protein concentration was assayed by the BCA method. The extracted or enriched protein samples were separated by denaturing 10% SDS-polyacrylamide gel electrophoresis and transferred into polyvinylidene fluoride membranes. After being blocked by 5% bovine serum albumin (BSA) for 120 min at room temperature, the membranes were incubated with primary antibodies from abcam and Cell Signaling Technology against GAPDH (ab8245, 1:8,000), mTOR (ab2732, 1:1,500), AKT (ab18785, 1:1,000), p-mTOR (ab109268, 1:1,500), p-AKT (ab38449, 1:1,000), P70S6K (#2708, 1:1,000), p-P70S6K (#9205, 1:1,000), and CAMK1D (ab172618, 1:1,500) overnight at 4°C. After washing three times, the blots were further incubated with goat anti-rabbit IRDye 800CW preadsorbed secondary antibody (1:10,000; Abcam; ab216773). The images were detected with an Odyssey infrared imaging scanner (LI-COR, USA).

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.