Deptor

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).

The rabbit polyclonal antibodies used were: Deptor (Upstate/Millipore, 09-463) I.F. 1:100; ERLIN2 (Sigma) WB 1:100; BiP (Cell Signaling, clone C50B12, #9956) WB 1:1000; PDI (Cell Signaling, clone C81H6, #9956) WB 1:1000; DERL3 (GeneTex, N3C2) WB 1: 1000; CKAP4 (GeneTex, N3C3) WB 1:1000; KEAP1 (Santa Cruz Biotechnology, clone E-20, sc-15246) WB 1:500; S6 p-S235/236 (Cell Signaling, #4858, clone D57.2.2E) WB 1:1000; Histone H3 (Abcam, clone ab1791) WB 1:1000; Cleaved caspase 7 (Cell Signaling, #9491) WB 1:1000; ATF4 (Santa Cruz Biotechnology, clone C-20, sc-200) WB 1:100. Mouse monoclonal antibodies: Deptor (Santa Cruz Biotechnology, clone A-3, sc-398169) WB 1:1000; β-actin (Sigma, clone AC-15, A5441) WB 1:10000; CHOP/GADD153 (Proteintech, clone 4D5A9) WB 1:500; S6 (Cell Signaling, clone 54D2) WB 1:1000; α-Tubulin (Calbiochem, clone DM1A) WB 1:1000; Flag (Sigma, clone M2, F1804) WB 1:1000. Secondary antibodies used were goat anti-mouse and goat anti-rabbit, conjugated to horseradish peroxidase (Biorad). Immunostained bands were detected by the chemiluminescent method (Pierce) and the images were acquired using Alliance system by UVITECH, Cambridge.