Tak228

Five human retinoblastoma cell lines, immortalized normal retinoblastoma pigmented epithelial cell line (RPE‐1), and normal human fibroblast BJ‐5ta were obtained from American Type Culture Collection or Chinese Academy of Sciences and were authenticated through short tandem repeat profiling analysis (Precision Biotechnology). Retinoblastoma and normal cells were maintained under the same culturing conditions as described in our previous studies.¹¹, ¹² Human primary retinal microvascular endothelial cell (HREC; Cell Systems) were cultured in basal M131 medium supplemented with microvascular growth supplement (Invitrogen). TAK‐228 (Selleckchem, 99% purity with HPLC) and MHY1485 (Selleckchem, 99% purity with HPLC) were reconstituted in dimethyl sulfoxide (DMSO), sterile‐filtered and stored in aliquots in −20°C. Antibodies against phospho‐Akt(Ser473), ‐mTOR(Ser2448), ‐S6K1(Thr389), ‐rS6(Ser240/244), ‐4EBP1(Thr37/460), ‐NDRG1(Ser330) and their corresponding total were all obtained from Cell Signaling Technology. Denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blot were performed using the standard protocol.¹³