Anti-mTOR antibody

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	Dilute primary Ab at 1:1000 and incubate overnight at 4°C

Protocol tips
Dilute primary Ab at 1:1000 and incubate overnight at 4°C

Publication protocol

Protein preparations and western blot analysis
Total protein was extracted from HepG2 cells using radioimmunoprecipitation assay cell lysis reagent containing proteinase and phosphatase inhibitors (Solarbio). The cells remained in the medium on ice for 30 min with re-dispersion every 5 min. Cell lysates were centrifuged at 12,000 x g for 10 min at 4°C, and the protein concentrations of the supernatants were determined using the BCA protein assay reagent kit (Beyotime). The supernatants containing total protein were mixed with a corresponding volume of 5X SDS loading buffer and were subsequently denatured by boiling for 10 min. Samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 5% stacking and 12% separating gels. Subsequently, the samples were transferred onto polyvinylidene difluoride (PVDF) membranes (0.2 μm, Immobilon-P; Millipore, Billerica, MA, USA) at 60 V for 2.5 h. After blocking with skimmed dry milk for 2 h at room temperature, the membranes were washed three times with Tris-buffered saline with Tween 20 (TBST) for 30 min. Then they were incubated overnight at 4°C with the appropriate primary antibody. The primary antibodies and dilutions used were as follows: rabbit anti-human OX1R (cat. no. ab68718, 1:1000), rabbit anti-human OX2R (cat. no. ab129432, 1:500), rabbit anti-human Akt (cat. no. ab8805, 1: 1,000), rabbit anti-human phospho-Akt (cat. no. ab8932, 1: 1,000), rabbit anti-human mTOR (cat. no. ab2732, 1: 2,000), rabbit anti-human phospho-mTOR (cat. no. ab109268, 1: 1,000), rabbit anti-human HIF-1α (cat. no. NB100-479, 1:1000), rabbit anti-human GLUT1 (cat. no. ab115730, 1: 1,000), rabbit anti-human β-actin (cat. no. ab8227, 1: 1,000). The membranes were washed and incubated at room temperature for 1.5 h with the secondary goat anti-rabbit lgG (H+L) antibody (Beyotime, cat. no. A0208, 1: 2,000) conjugated with horseradish peroxidase, then washed three times with TBST for 30 min. The membranes were subjected to enhanced chemiluminescence (ECL) using an ECL detection kit (Beyotime, cat. no. P0018) and quantified using Quantity One software (Bio-Rad Laboratories Inc., Hercules, CA, USA).

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