Nitrocellulose membrane

Liver tissue samples were homogenized with 5–10 volumes of lysis buffer (Sigma-Aldrich, USA) containing 1 mM phenylmethanesulfonyl fluoride (PMSF) and 1X protease inhibitor cocktail (Sigma-Aldrich, USA). The homogenate was centrifuged at 10836 ×g for 10 min and supernatants were used as the whole protein extract. Total protein was estimated using Bradford method. About 30 μg of the liver protein was separated by 10% SDS-PAGE and then transferred onto a nitrocellulose membrane (0.45 μm, Bio Basic, Inc.) with miniprotein two-gel electrophoresis system (Bio-Rad, USA). The transferred membrane was blocked through incubation with 5% bull serum albumin (BSA) for 3 h at room temperature. The transferred membranes were then blotted with the following primary antibodies at 4°C overnight at dilution of 1 : 1000: phosphor-mTOR (P-mTOR), total-mTOR (T-mTOR), P-AKT, T-AKT, P-AMPK, T-AMPK, P-ERKs, T-ERKs, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Beverly, MA), followed by treatment with horseradish peroxidase-conjugated secondary antibodies diluted 1 : 2000 (Santa Cruz, USA). Immunoreactive bands were visualized with an ECL detection system (GE Healthcare, UK). The intensity of the bands was quantified by scanning densitometry using software Quantity One-4.5.0.

A part of liver tissue of each experimental mouse was homogenized in eight volumes of radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, USA) which contains 1 mM Phenylmethanesulfonyl fluoride (PMSF) and 1x protease inhibitor cocktail (Sigma-Aldrich, USA). The protein concentration was detected by Bradford method, and 30 μg of sample was separated by 10% SDS-PAGE and then transferred onto a nitrocellulose membrane (0.45 μm, Bio Basic, Inc.) with the Mini-Protean two-gel electrophoresis system (Bio-Rad, USA). The blocked membranes with target strips were incubated with primary antibodies at 4°C overnight at dilution of 1 : 1000 as follows: phosphor-AMPK (P-AMPK), total-AMPK (T-AMPK), peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α), phosphofructokinase-1 (PFK-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Beverly, MA), followed by blotting with horseradish peroxidase-conjugated secondary antibodies diluted 1 : 2000 (Santa Cruz, USA). After visualizing with ECL detection system (GE Healthcare, UK), the intensity of target bands were quantified by using ImageJ.

The treated cells were lysed with RIPA buffer containing 1% protease inhibitor cocktails and 2% phenylmethanesulfonyl fluoride (PMSF) (all from Sigma-Aldrich). For the detection of ERK translocation, the preparation of cytoplasmic and nuclear extracts was carried out as described in a previous study by Yang et al (21 (link)). After the supernatant collection, the protein concentration was determined using the Bradford method. Proteins were separated on a 10% SDS-PAGE gel and transferred electrophoretically onto nitrocellulose membranes (Bio Basic, Inc., Markham, Ontario, Canada). The transferred membranes were then blotted with primary antibodies (dilution of 1:1,000) to: phosphorylated ERK (p-ERKs), total ERK (t-ERKs), phosphorylated AKT (p-AKT), total AKT (t-AKT), cleaved poly(ADP-ribose) polymerase (PARP), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and lamin B (Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight, followed by treatment with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Chemiluminescence was detected using ECL detection kits (GE Healthcare, Buckinghamshire, UK). The intensity of the bands was quantified by scanning densitometry using Quantity One 4.5.0 software (Bio Basic, Inc.).