Mouse anti mtor

Nitrocellulose membranes containing the transferred proteins were blocked in Intercept Blocking Buffer (LI-COR Bioscience) for 1-hour at room temperature. Membranes were probed with primary antibodies, including mouse anti-mTOR (1:1000; Cat# 4517), mouse anti-Akt (1:1000; Cat# 2920), mouse anti-GSK3β (1:1000; Cat# 9832), mouse anti-ERK (1:1000; Cat# 9107), rabbit anti-phospho mTOR (1:1000; Cat# 5536 S), rabbit anti-phospho Akt (1:1000; Cat# 4060 S), rabbit anti-phospho GSK3β (1:1000; Cat# 5558), rabbit anti-phospho ERK (1:1000; Cat# 4376) (all antibodies were purchased from Cell Signaling Technologies) diluted in Intercept Blocking Buffer + 0.2% Tween-20 and incubated overnight at 4 °C, before being exposed to secondary antibody (IRDye^® 680RD Donkey anti-Mouse IgG and IRDye^® 800CW Donkey anti-Rabbit IgG) (LI-COR Biosciences) for 1-hour at room temperature in the dark.
Membranes were scanned on the LI-COR Bioscience Odyssey CLx imaging system and imaged using LI-COR Image Studio software version 2.1.10. All densitometry analyses were performed using Image Studio Light version 5. The region of interest encircling each band was defined automatically. All bands at the correct molecular weight were analyzed as the signal for that target protein. Values for each protein were normalized to loading control β-tubulin (Abcam).

BEZ235, TSA and 3-MA were purchased from Selleck company (Selleck, Shanghai, CHINA). Antibodies for western blotting and flow cytometry were: mouse anti-cleaved caspase-3, mouse anti-cleaved caspase-8, mouse anti-cleaved caspase-9, mouse anti-p-Akt, mouse anti-Beclin-1, mouse anti-LC3, mouse anti-S6, mouse anti-p-S6, mouse anti-4EBP1, mouse anti-p-4EBP1, mouse anti-mTOR (Cell Signaling, USA), mouse anti-PARP-1 (Abcam, USA).