Ab179463

Cells and ventricular tissue were lysed with RIPA buffer (Sigma–Aldrich, USA), and the protein concentrations of the lysates were determined using BCA Protein Assay Reagent (Pierce Biotechnology, USA). Total protein samples (40 μg) were separated electrophoretically, transferred to a PVDF membrane and probed using monoclonal primary antibodies against PI3K (1:5000, Abcam, ab139307), p-PI3K (1:1000, Abcam, ab32089), AKT (1:1000, Abcam, ab179463), p-AKT (1:1000, Cell signaling, 4060s), KDM5A (1:5000, Abcam, ab194286), IGF1 (1:1000, Abcam, ab223567), MYH11 (1:1000, Abcam, ab82541), TGFB3 (1:1000, Abcam, ab15537), and β-actin (1:500, Abcam, ab5694), followed by an HRP-conjugated secondary antibody (1:5000; Abbiotec, USA). Bands were exposed using an ECL kit (Bio–Rad, USA) and analyzed using Image-Pro Plus software. LY294002 (20 μmol/L, Cell signaling, 9901s) is an inhibitor of PI3K, and ARQ-092 (10 μmol/L, Abcam, ab235550) is an inhibitor of AKT.

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.