Detection of NF-κB p65 protein expression by western blot analysis

At the end of T3, pentobarbital sodium (150 mg/kg body weight) was injected intraperitoneally until breathing and heartbeat stopped. The right hippocampus tissues (20 mg) were isolated from the rats, mixed with 100-200 µl lysates and homogenized in a glass homogenator at 4˚C for 15 min, 1.2x10⁴ rpm. The supernatant was taken. We used the BCA kit (Abcam) to detect protein concentration. SDS-PAGE electrophoresis was used to distinguish the proteins before transferring to the nitrocellulose membrane, and then the proteins were blocked at room temperature for 1 h with 5% PBS solution. Next, NF-κB p65 (1:1,000) was added and maintained overnight at 4˚C. Membranes were washed with PBS solution. This operation was repeated 3 times before a secondary antibody (HRP cross-linking, 1:10,000) was added, and the mixture was allowed to stand at room temperature for 1 h. The membrane was finally washed with PBS solution and protein bands were visualized using an electrochemiluminescent substrate kit (cat. no. ab133406; Abcam). The internal reference protein was β-actin. The relative protein expression of NF-κB p65 was calculated: Relative expression level of protein=(gray value of protein band)/(gray value of β-actin band). We used ImageJ (National Institutes of Health) to measure the gray value.