Ab9726

To evaluate the level of protein expression in the brain, the brain tissues were denatured by boiling for 10 min. Then, the samples were separated by 10% polyacrylamide gel electrophoresis and transferred onto PVDF membranes. After blocking in 5% skim milk for 1 h, the membranes were probed with primary antibodies such as GR (1:1000; MA1-510, Invitrogen), p-GR (1:1000; 4161s, Cell Signaling), CREB (1:1000; ab31387, Abcam), p-CREB (1:1000; ab32096, Abcam), BDNF (1:1000; ab108319, Abcam), 5-HT_1AR (1:1000; ab85615, Abcam), 5-HTT (1:800; ab9726, Abcam), α-tubulin (1:1000; ab7291, Abcam), and β-actin (1:1000; MA5-11869, Thermo-Fisher Scientific) antibodies overnight at 4 °C. The membranes were washed 3 times and incubated with an HRP-conjugated anti-rabbit (1:5000; for p-CREB, CREB, BDNF, 5-HT_1AR, and 5-HTT) or anti-mouse antibody (1:5000; for p-GR, GR, α-tubulin, and β-actin) for 45 min. The proteins were visualized using an ECL Advanced Kit. Protein expression was observed using the FUSION Solo System (Vilber Lourmat, Collegien, France), and band intensity was analyzed with ImageJ version 1.46 (NIH, Bethesda, MD, USA).