Phosphor mtor ser 2448

Briefly, cells were washed with ice cold PBS and lysed with ice-cold phospholysis buffer supplemented with protease and phosphatase inhibitors. After lysis, the suspension was centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant was collected and stored for further processing. The total protein content of the extracted whole cell lysates were determined by the Bradford method, and 50μg of the total extracted protein from respective samples were denatured and subjected to immunoblotting. The following primary antibodies and their respective secondary antibodies labeled with HRP were used for detecting the expression levels of various proteins with enhanced chemiluminescence (Pierce ECL substrate, USA). Primary antibodies used were DEPTOR, eNOS, iNOS, HPV E6, HPV E7, HSC-70 (Santa Cruz, USA); mTOR, phosphor-mTOR (ser 2448), phospho-pERK1/2, phospho-p38, phospho-4EBP1, phospho-S6K, AKT, phospho-AKT, PARP, Caspase 3, Caspase 9, Caspase 7, PUMA, p53 (Cell Signaling Technology, USA); pRb (Oncogene, USA); phospho-IRS1, phospho-GSK3β (Pierce, USA); Vinculin, anti-rabbit HRP, anti-goat HRP, anti-mouse HRP antibodies (Sigma, Germany).