Bca kit

The total protein was extracted with RIPA buffer (containing 0.1% SDS, 1% protease inhibitor, and 1% phosphatase inhibitor; Solarbio, R0020). Protein quantification was performed using the BCA Kit (Coolaber, SK1070-5000T). 20 μg protein lysis was prepared for SDS-PAGE (6% concentrated gel and 12% and 15% separated gel) and transferred to 0.22 μm Immobilon-PSQ PVDF membrane (Merck Millipore Ltd., ISEQ00010). The membranes were blocked with 5% nonfat-dried milk (Coolaber, CN7861-500G) at room temperature for 2 h, washed 3 times with TBST (Solarbio, No. T1080) for 15 minutes each time, and hatched with primary antibody at 4°C overnight. After being washed, the membranes were hatched with the corresponding secondary antibody at room temperature for 1 h and then washed 3 times with TBST for 15 minutes each time. β-actin was used as an internal control. All immunoreactive protein bands were visualized by BeyoECL Moon (Beyotime, P0018FM-2) and quantified by software Image-Pro Plus 6.0.

Brain samples were homogenized and transferred to a separate tube containing RIPA buffer for 35 min on ice. After centrifugation (13,000 rpm, 30 min, 4°C), protein concentrations were measured in the resulting supernatants using a BCA kit (Coolaber, Beijing, China). Protein lysates (20 μg per well) were separated by sodium dodecyl sulfate-polyacrylamide gel (10%) electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking in 5% skimmed milk for 2 h at room temperature on a shaking platform, the membranes were incubated overnight at 4°C with primary antibodies for the detection of GLUT1 (1:8,000; Abcam, Cambridge, MA, United States), PFK (1:1,000; Abcam, Cambridge, MA, United States), GLUT5 (1:500; Affinity Biosciences, Cincinnati, OH, United States), KHK (1:2,500; Abcam, Cambridge, MA, United States) and β-actin (1:8,000; ABclonal, Wuhan, China). The membranes were then washed and incubated with the secondary detection antibody (1:3,000; horseradish peroxidase-conjugated anti-rabbit IgG; ABclonal, Wuhan, China). Immunoreactive protein bands were visualized using an ECL Western blot substrate (Pierce, Rockford, IL, United States) and images were captured on an Alpha Innotech Chemilmager (Alpha Innotech, San Leandro, CA, United States). Quantification of the proteins was carried out using Image J Software.