Anti ikbα

Protein expression in brain tissues was detected by western blotting. Fifty milligram protein was extracted from 100 mg brain tissue samples and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to standard methods. Then, proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Germany) and blocked using 5% skim milk. The membranes were incubated overnight at 4°C with the following antibodies: anti-cleaved caspase-3, anti-IkBα (Proteintech, Wuhan, China), anti-phosphor-IkBα (Abscitech, Shanghai, China), anti-acetyl-histone H3, anti-acetyl histone H4, anti-NF-κB p65 (Cell Signaling Technology, Danvers, United States), and anti-GAPDH (Cell Signaling Technology, Danvers, United States) as the control, and then incubated with a secondary antibody for 2 h at room temperature. The membranes were visualized with an enhanced chemiluminescence (ECL) western blotting detection system (Amersham, United States). The changes in the protein levels were calculated using ImageJ software (Patel et al., 2011 (link)).

Western blotting analysis was performed as described in the previous report (Zhao et al., 2021 (link)). The cells were lysed in lysis buffer. After centrifugation, the supernatant was collected and the protein concentration was quantified by BCA assay kit. The samples were boiled at 95°C for 5 min and immediately frozen in a –80°C refrigerator. The primary antibodies were used as follows: anti-CD36 (ABclonal, Wuhan, China, A5792, 1:1,000), anti-GAPDH (ABclonal, Wuhan, China, AC033, 1:1,000), anti-FTO (ABclonal, Wuhan, China, A1438, 1:1,000), anti-P65-S536 (ABclonal, Wuhan, China, AP0475, 1:1,000), anti-IKB-α (Proteintech, Wuhan, China, 10268-1-AP, 1: 1,000), and anti-β-actin (ABclonal, Wuhan, China, AC026, 1: 1,000).