P s6 s235 s236

Cells were lysed in RIPA buffer, sonicated at 4°C a nd centrifuged at 12,000g for 15 minutes. Proteins were separated on 4%–20% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Amersham), blocked in Odyssey Blocking Buffer 1X (LI-COR Bioscience) for 60 minutes at room temperature and incubated overnight with primary antibodies diluted in Odyssey Blocking Buffer 1:1 with phosphate-buffered saline-PBS and 0.1% Tween 20. Antibodies for RICTOR (1:1000), ULK1 (1:1000), FOXO3 (1:1000), ATG14 (1:1000), P-AKT (Ser473) (1:2000), AKT (1:1000), ATG5 (1:1000) ATG3 (1:1000), P-S6 (S235/S236) (1:2000), S6 (1:1000), LC3A/B (1:1000) were purchased from Cell Signaling. Antibody for Gabarapl1 (1:500) was purchased from Fisher Scientific and that for β-actin (1:5000) from Abcam. After three washes with Tris-buffered saline–0.1% Tween (TBST), membranes were incubated with IR Dye labelled secondary antibody (goat anti-Mouse 1:10000 and goat anti-Rabbit 1:5000, LI-COR Biosciences) diluted in Odyssey Blocking Buffer 1:1 with PBS, 0.1% Tween 20 and 0.01% SDS. Subsequently, the membranes were washed 3 times with TBST, kept in water and visualized by Odyssey Scanner (LI-COR Biosciences). Densitometry was performed using LI-COR Odyssey Software Version 4.0 (LI-COR Biosciences).