Anti acetyl histone h4

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)).

Cell total proteins were extracted using RIPA buffer and the protein concentrations were determined by a BCA kit (Thermo, USA). 20 μg of total lysates were separated by a 10% SDS-PAGE gel and transferred onto a PVDF membrane and blotted with 5% bovine serum albumin (BSA) in TBS for 90 minutes, and then incubated with primary antibodies of anti-acetyl-lysine, anti-acetyl histone H4, and anti-total histone H4 (Cell Signaling Technology, USA) overnight at 4°C. After washing, the membrane was incubated with HRP conjugated horse anti-rabbit antibody, 60 minutes at room temperature. The membranes were visualized by ECL Plus western blotting detection reagents (Millipore, USA). Total histone H4 was used as an internal control. The gel was stained with Coomassie Brilliant Blue G250.