Phospho pi3 kinase p85

Celastrol and low molecular weight heparin (LMWH) were purchased from Dalian Meilun biotechnology company. Dimethylaminopyridine (DMAP), N-Hydroxysuccinimide (NHS) and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were obtained from Sigma–Aldrich. Dulbecco's modified Eagle's medium was purchased from Gibco. PI3 Kinase p85α, Phospho-PI3 Kinase p85, Phospho-Akt, anti-Akt, LC3B, P62, mTOR, Phospho-mTOR, and Cleaved Caspase-3 (Asp175) antibodies were purchased from cell signaling technology. Anti-β-actin was purchased from Sigma–Aldrich. Anti-myeloperoxidase was purchased from Abcam. P-selectin and Anti-RIP3 was purchased from Santa Cruz.

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (CWBIO, China) with protease and phosphatase inhibitors (CWBIO, China). An equal amount of total protein lysates (30 ng) was separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane and then incubated with primary antibodies specific for AGO1 (1:1,000 dilution; Proteintech, USA), anti-PI3Kinase p85 (1:1,000 dilution; Cell Signaling Technology, USA), phospho-PI3Kinase p85 (1:1,000 dilution; Cell Signaling Technology, USA), anti-AKT kinase (1:1,000 dilution; Cell Signaling Technology, USA), phospho-AKT (Ser 473, 1:1,000 dilution; Cell Signaling Technology, USA), anti-mTOR (1:1,000 dilution; Cell Signaling Technology, USA), phospho-mTOR (1:1,000 dilution; Cell Signaling Technology, USA), and GAPDH (1:1,000 dilution; Proteintech, China) at 4°C overnight, followed by incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 2 h. Bands were visualized using Immobilon ECL substrate kit (Millipore, Germany), and the images were captured via Bio Spectrum 600 Imaging System (UVP, USA).