Phosphor akt

The protein sample of each liver with 200 mg was prepared and protein concentration was measured as described in a previous study (Tang et al., 2015 (link)). One hundred microgram protein of each sample was loaded onto the 10% SDS-PAGE gel and run at 120 V for 60 min. Subsequently, the protein on the gel was transferred to polyvinylidenedifluoride membranes. The membrane was blocked with 5% non-fat milk in TBS for 60 min, incubated with first antibodies of IGF-1 receptor, phosphor-IGF-1 receptor, AKT (Cell Signaling Technology, Danvers, MA, United States), phosphor-AKT (Abcam, Hong Kong, China), and GAPDH (Beijing ComWin Biotechnology, Beijing, China) in 2% non-fat milk overnight at 4°C, respectively. Afterward, the membrane was incubated with a secondary antibody (Beijing ComWin Biotechnology, Beijing, China) for 45 min after washing three times with TBS. The bands were developed using enhanced chemiluminescent reaction (Beijing ComWin Biotechnology, Beijing, China), and density analysis was performed using Image J software (National Institute of Health, Rockville, MD, United States).

One part of the kidney tissue was homogenized in a radioimmunoprecipitation assay buffer (RIPA; Sigma-Aldrich, USA) containing 1% protease inhibitor cocktail and 2% phenylmethanesulfonyl fluoride (Sigma-Aldrich, USA). Protein concentrations were determined by the Bradford method, and 40 μg proteins were separated using 10% SDS-PAGE gel and transferred electrophoretically onto nitrocellulose membranes (0.45 μm; Bio Basic, Inc., USA). The transferred membranes were blotted with primary antibodies at 4°C overnight at a dilution of 1 : 1000: phosphor-AKT (ab131443), total-AKT (ab200195), phosphor-GSK-3β (ab75745), total-GSK-3β (#32391), and glyceraldehyde-3-phosphate dehydrogenase (#2118) (Abcam, Cambridge, UK) and then incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, USA). Chemiluminescence was detected using ECL detection kits (GE Healthcare, UK). The intensity of the bands was quantified by scanning densitometry using Image J software (National Institutes of Health, Bethesda, USA).