Anti phospho 4ebp1 thr70

Cells were collected after stimulated with 100 μM FAC or 100 μM DFO for 24 h. Cellular proteins were extracted by RIPA lysis buffer containing protease and phosphatase inhibitors. SDS-PAGE was used to separate the proteins. After running process, gels were transferred to PVDF membranes and immersed in primary antibodies. The next day, membranes were incubated with secondary antibodies and be visualized by chemiluminescence detection kit (Beyotime). Slc25a28 antibody (ab90170, 1:100) was from Abcam. antibodies specific for SLC25A37/Mitoferrin1 (26469-1-AP, 1:100) and Glut1 (66290-1-Ig, 1:100) were purchased from Proteintech. Anti-phospho-AMPK (Thr172) antibody (#2535S, 1:100), Anti-AMPKα Antibody (#2532, 1:100), anti-p70-S6K (9202S, 1:100), anti-phospo-p70-S6K (Thr389) (9234S, 1:100), anti-Hexokinase 2 (2867S, 1:100), anti-phospho-4EBP1 (Thr70) (13396, 1:100) and anti-4EBP1 (9644s, 1:100) were from Cell Signaling Technology. Anti-PCNA (2586S, 1:100) was from Cell Signaling Technology. The anti-GAPDH antibody (BM1623, 1:1000), anti-β-actin antibody (BM0627, 1:1000), anti-α-tubulin antibody (BM1452, 1:1000), anti-rabbit IgG-HRP antibody (BA1054, 1:5000), and anti-mouse IgG-HRP antibody (BA1050, 1:5000) were purchased from Boster Biological Technology (Wuhan, China).

Human cervical carcinoma cell lines (CaSki and SiHa) and human embryonic kidney 293T (HEK293T) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in monolayer cultures according to ATCC recommendations. HUVECs were purchased from Clonetics (Walkersville, MD, USA), and seeded on 0.3% gelatin-coated dishes (Sigma, St. Louis, MO, USA) using the EGM-2 BulletKit medium (Clonetics). Wortmannin and LY294002, which are PI3K inhibitors, were obtained from Sigma (St. Louis, MO, USA). The following antibodies were used in this study: anti-HPV16 E6 (ab226447), anti-IgG (Abcam, Cambridge, UK), anti-IRF-1, anti-Flag, anti-cyclin D1, anti-CDK4, anti-NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Bax, anti-Bcl-xL, anti-Bcl-2, anti-p53, anti-VEGFR-1, anti-VEGFR-2, anti-phospho-VEGFR-2(Tyr-1175), anti-HIF-1α, anti-PI3K, anti-phospho-PI3K, anti-Akt, anti-phospho-Akt(Ser473), anti-PDK-1, anti-phospho-PDK-1(Ser241), anti-mTOR, anti-phospho-mTOR(Ser2448), anti-4E-BP1, anti-phospho-4E-BP1(Thr70) (Cell Signalling, Beverly, MA, USA), anti-p21, anti-VEGF (Ab-1; Oncogene, Cambridge, MA, USA), and anti-β-actin (Sigma, St. Louis, MO, USA).