P70s6k

Whole cells were lysed by lysis buffer (RIPA buffer contains protease inhibitors and phosphatase inhibitors). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific, Shanghai, China). A total of 30 μg of protein was separated on SDS-PAGE gels (mTOR/P-mTOR 6% gel, pAKT473/AKT/β-Actin 12% gel, pp70s6k/p70s6k 10% gel) and transferred onto Immobilon-P membrane (IPVH07850; Millipore, Darmstadt, Germany). Membranes were first blocked with 5% non-fat milk in Tris-buffered saline with 1% Tween 20 (TBST) at room temperature for 1 h, then incubated overnight at 4 °C with the following primary antibodies: pAKT473 (Cell Signaling Technology, Boston, MA, USA), pAKT308 (Cell Signaling Technology, Boston, MA, USA), AKT (Cell Signaling Technology, Boston, MA, USA), pmTOR (Cell Signaling Technology, Boston, MA, USA), mTOR (Cell Signaling Technology, Boston, MA, USA), pp70s6k (Cell Signaling Technology, Boston, MA, USA), and p70s6k (Cell Signaling Technology, Boston, MA, USA), diluted at a 1:1000 ratio in TBST with 3% bovine serum albumin (BSA), washed with TBST, and finally incubated with HRP-labelled goat anti-mouse or anti-rabbit IgG secondary antibodies at room temperature for 1.5 h. The proteins were visualized by Plus-enhanced chemiluminescence using Universal Hood III (Bio-Rad, Hercules, CA, USA).

Antibodies were obtained from Cell Signaling, Boston, MA, USA (Smad2 #5339, pERK1/2^T202/Y204, ERK1/2, pFoxO3a^T32, p-mTOR^S2448, mTOR, rictor, raptor, GβL, p4E-BP1^S65, 4E-BP1, pAkt^S473, Akt, ATG7, LC3b, SGK, pULK1^S757), Novus Biologicals, Littleton, CO, USA (eIf3f Lamp2), Epitomics, Burlingame, CA, USA (pSmad3^S423/S425, Smad3), Invitrogen, Grand Island, NY, USA (pSmad2^S465/S467), Abcam, Cambridge, MA, USA (p70S6k, p-p70S6k^T389, Myogenin, FoxO3a, p62, ULK1), Millipore, Billerica, MA, USA (pFoxO3a^S252, p21), ECM Biosciences, Versailles, KY, USA (atrogin-1), and Santa Cruz Biotechnology, Dallas, TX, USA (GAPDH). Secondary antibodies were obtained from GE Healthcare, Pittsburgh, PA, USA (anti-rabbit, anti-mouse, anti-rat) and Sigma-Aldrich, St Louis, MO, USA (anti-goat).

P-PDK1(Ser241), Akt, p-Akt(Ser473), 4EBP1 and p-4EBP1(Ser65) and α-tubulin were purchased fromCell Signaling Technology (Danvers, MA, USA). P70S6K and p-P70S6K(Thr470) were acquired from Millipore (Boston, MA, USA). Antibodies against LC3, SQSTM1, Atg5, Beclin1, LAMP1, Bcl-2, Bax, cytochrome C, active caspase-9, active caspase-3, cleaved PARP, mTOR, p-mTOR(S2448) and β-actin were obtained from Abcam (Cambridge, UK). YZT was dissolved in DMSO and diluted with culture medium. CQ, 3-methyladenine (3-MA) and rapamycin (RAPA) were bought from Sigma-Aldrich (Saint Louis, MO, USA).