Ace h3

Western blotting was used to determine the quantity of target proteins being made in the ischemic hemisphere. Briefly, brain tissues were milled in liquid nitrogen, and proteins were extracted using RIPA cleavage buffer. Protein intensity, with BSA as a standard, was measured according to the Bradford method. Each sample was separated using 12% SDS-PAGE and transferred onto a methanol-activated PVDF membrane (Millipore, Burlington, MA, USA) by wet electrotransfer. The membranes were blocked for 1.5 h at room temperature with 5% BSA. The membranes were then incubated with primary antibodies against Ace-H3 (Millipore, cat #9717, 1:1000), H3K9ace (Cell Signaling Technology, cat #6949, 1:1000), H3K27ace (Cell Signaling Technology, cat #4353, 1:1000), Histone3 (Abcam, ab1791, 1:1000), Bcl-2 (Abcam, ab59348, 1:1000), Bax (Cell Signaling Technology, cat #2772, 1:1000), caspase-3 (Cell Signaling, cat #9662, 1:1000), cleaved caspase-3 (Cell Signaling, cat #9664S, 1:1000) and GAPDH (SAB, cat #21612S; 1:1000). The total amount of ECL liquid was absorbed in a 1:1 ratio (solution A: B) to uniformly cover the entire film and was observed using an AI600 imaging system (GE Healthcare, USA). GAPDH was used as an internal reference for comparison of grayscale values.