Rps6kb1

Briefly, the mice organs were collected and disrupted in lysis buffer containing protease inhibitors (Roche Diagnostics, Berlin, Germany). After centrifugation, the supernatant was collected, and equivalent amounts of protein were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The protein bands were visualized using DyLight 800/DyLight 680-conjugated secondary antibodies, and an infrared fluorescence image was obtained using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Western blot analyses were performed by ImageJ with anti-Gapdh (KM9002, Sungene, Tianjin, China), anti-Lc3b (SAB4200361, Sigma, St Louis, MO, USA), anti-Sqstm1 (PM045, MBL International, Japan), and anti-Eva1a (NB110-74787, Novusbio, Littleton, CO, USA) antibodies. Antibodies against Ulk1, Akt, Mtor, Erk1/2, Lkb1, Ampk, Rps6kb1, and Eif4ebp1 and against phosphorylated Ulk1, Akt, Mtor, Erk1/2, Lkb1, Ampk, Rps6kb1, and Eif4ebp1 were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against Drp1, Tomm20, Pink1, Parkin, Bnip3, Mitofusin2, and Pgc1 were purchased from Abcam (Cambridge, UK). DyLight 800/DyLight 680-conjugated secondary antibodies against mouse or rabbit IgG were purchased from Rockland Immunochemicals (Limerick, PA, USA).

Breast cancer cells were lysed in lysis buffer (2 g SDS, 1.55 g DTT, 6 ml Tris (1 M, pH 6.8), 10 ml glycerol, and ddH₂O up to 100 ml). Fifty to eighty micrograms of the total protein was separated on an SDS/PAGE (12% gel), and then was transferred on to PVDF membrane (Millipore). The membranes were blocked with 5% skim milk dissolved in PBS and Tween 20 (PBST) for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. After incubating with horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h, the immune activity on the membranes was detected with ECL-Plus kit (Amersham Biosciences). Antibody against eukaryotic translation initiation factor 4E (eIF4E) (#2067), AMPKα (#5832), and ribosomal protein S6 kinase B1 (RPS6KB1) (#2708) were purchased from Cell Signaling Technology. Antibody against GAPDH was purchased from Santa Cruz Biotechnology (SC-32233). All the secondary antibodies were from Santa Cruz Biotechnology.