Western Blot Analysis of PDXK Protein

The Western blot experiment was carried out as previously described (Liu et al., 2014 (link)). In brief, tissues from the brain cortex were dissociated and homogenized by radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor cocktail (Beyotime, Jiangsu, China). A volume of about 80 μg proteins was resolved in 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel (Beijing Junyi Electrophoresis Co., Ltd., Beijing, China) with electrophoresis buffer at 60 V for 30 min, then 100 V for 90 min. The proteins bands were transferred to a polyvinylidenedifluoride (PVDF) membrane with 300 mA for 40 min. For blocking, the membrane was incubated in 5% skim milk at room temperature for 1 h and then was incubated with the primary antibody of PDXK (mouse, Abcam, 1:2,000) and β-actin (mouse, Abcam, 1:5,000) at 4°C overnight. The next day, the membrane was rinsed with 1 × TBS containing 0.2% Tween-20 (TBST; Shanghai Double-Helix Biotech Co., Ltd., Shanghai, China) for four times. Then, the membrane was incubated with 1:5,000 secondary antibody (goat antimouse IgG, ZSGB-BIO, China). Finally, the membrane was rinsed in TBST for four times, and the immune complexes were developed with enhanced chemiluminescence reagent and visualized by ChemiDoc XRS System with Image Lab Software 2.0 (Bio-Rad Laboratories, Inc., Hercules, CA, United States).