Mtorc1

After centrifugation (10,000 g, 15 min at 4°C), the supernatants were collected for protein analysis. The protein concentration was determined by Bradford protein assay. Western blot analysis was performed as previously described. Briefly, 50 µg proteins from each sample were loaded onto an SDS polyacrylamide gel for electrophoresis and subsequently transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat dry milk in PBS for an hour and incubated with a primary antibody against BMAL1 (1:200 dilution), AKT (1:1000 dilution), p-AKT (Thr 308) (1:2000 dilution), mTORC1 (1:1000 dilution), p-mTORC1 (Ser2481) (1:2000 dilution), S6K1 (1:1000 dilution), p70-S6K1 (Thr389) (1:2000 dilution), NfκBp65 (dilution 1:250 dilution), p-NfκB (Ser536) (1:250 dilution) overnight at 4°C (BMAL1, mTORC1, p-mTORC1, NfκBp65 and p-NfκB were purchased from Abcam; AKT, p-AKT, S6K1S and p70-S6K1 were purchase from CST Danvers, USA). Samples were then incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, USA) (1:5000 dilution). Band intensities were visualized with chemiluminescence reagent (Millipore Corp.) by the BioMax film (Kodak) and then analyzed with Gel-Pro analyzer 4.0 software. β-Actin (Santa Cruz, USA) protein was utilized as a loading control.

Hypothalamus was mixed with RIPA buffer (RIPA : Cocktail = 100 : 1),and centrifuged at 4°C, 12000 rpm for 10 min; supernatants were boiled for 5 minutes with 5x SDS-PAGE Loading (volume 4 : 1). SDS-PAGE was prepared with deionized water, 1.5 M Tris-HCl (PH 8.8), 10% SDS, 10% APS and TEMED. Protein samples (100 μg) were transferred to a nitrocellulose membrane (Hybond, USA) with 200 V voltage for 2 hr and blocked with 5% 5% BSA for 2 hr and then washed with TBST 3 times, 5 min/time. After blocking, the membrane was incubated with the primary antibody (mTORC1 : 1 : 1000, abcam, UK) overnight at 4°C. After incubation, the membranes were washed 3 times, 5 min/time, followed by incubation with the secondary antibody (1 : 5000) for 2 hr and washed 3 times, 10 min/time. Protein bands were analyzed using Gel image analysis and imaging system (JunYi, Beijing, China).