T pi3k

Cells were lysed by RIPA. After being shocked and ice-bath for 30 min, cells were centrifuged for 20 min at 22,000 rpm at 4°C. The supernatants were collected. Cell membrane protein and cytoplasmic protein were separated using Mem-PER^TM Plus Membrane Protein Extraction Kit (Thermo). The concentration of protein was accessed using BCA. 20–40 μg protein was separated by 8–12.5% SDS-PAGE, then transferred to polyvinylidene fluoride membrane (Immobilon-P) using Trans-Blot Turbo Transfer System (Bio-Rad). DMT1, IRP1, IRP2, TfR1, TfR2, ferritin, T-PI3K, phospho-PI3K antibodies were obtained from Abcam. T-STAT3 (0.1 mg/ml), phospho-STAT3 tyr705 (1 mg/ml) were acquired from R&D. T-ERK, phospho-ERK, pan-AKT, phospho-AKT (1:1000) were obtained from Cell signaling technology. GAPDH and β-actin antibodies were purchased from Biotime. After being cultured with primary antibodies overnight at 4 °C, proteins were cultured with the HRP-labeled secondary antibody (KPL). Proteins were visualized using enhanced chemiluminescence (Pierce). All experiments were performed in triplicate.

Total protein was extracted using RIPA lysis buffer (Solarbio, Beijing, China) with the Protease Inhibitor Cocktail (Sigma) and qualified using BCA detecting kit (Tiangen, Beijing, China). All procedures were performed according to the manufacture’s protocol. Then, equal amounts of proteins were loaded in sodium dodecyl sulfate-polyacrylamide gels and transferred to a polyvinylidene fluoride membrane. After blocked with tris buffered saline-tween with 5% nonfat milk at room temperature (BD Bioscience, USA) for 0.5 hrs, the membranes were incubated with PTEN, t-PI3K, p-PI3K, t-AKT, p-AKT, Caspase3, Bcl-2, BAX and β-actin antibodies (dilution rates of 1:500) (Abcam, Cambridge, MA, USA) at 4 °C overnight. Then, the membranes were incubated with anti-rabbit or anti-mouse secondary antibodies (1:5000) (Solarbio, Beijing, China) after washed. Finally, the signals were detected by SuperSignal West Femto Trial Kit (Thermo Fisher Scientific, USA).