|The silenced cells were treated with or without the autophagy inhibitors BafA1 (Millipore, 196000) or CQ (Sigma-Aldrich, C6628) and then lysed in RIPA buffer (1% NP40, 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.25% sodium deoxycholate, 1% sodium dodecyl sulfate (SDS), protease inhibitor cocktail and phosphatase inhibitor). The proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes for immunoblotting. The membrane were rinsed with Odyssey Blocking Buffer (Li-COR, 927-50000) and then incubated overnight at 4°C with primary antibodies against ATG4B (A2981), MAP1LC3 (L7543) and ACTB (A5441) (purchased from Sigma-Aldrich); p27kip1 (purchased from Santa Cruz Biotechnology); and CCND1 (cyclin D1, 2926), pThr172-AMPK (5536), AMPK (2532), pSer792-raptor (2083) and raptor (2280) (purchased from Cell Signaling Technology). The proteins were probed with an HRP-labeled secondary antibody (Santa Cruz Biotechnology, sc-2004 or sc-2005) and detected with an ECL reagent. The membrane was scanned and analyzed for protein expression levels using the ChemiDoc XRS Imaging System (Bio-Rad). The protein levels were measured using ImageJ software.|
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