Anti cyto c

Fresh brain tissue around the hematoma was removed, and protein extraction of both the cytosolic and mitochondrial fractions was performed using a multiple centrifugation method.23 (link) Briefly, the homogenized tissue was centrifuged at 750× g for 10 minutes at 4°C and then at 10,000× g for 20 minutes. The pellets were used to obtain the mitochondrial fraction. Then the supernatant was further centrifuged at 100,000× g for 60 minutes and was used for the cytosolic analysis. After the protein concentrations were determined, equal amounts of the samples were loaded, and the proteins were separated by 10%–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The primary antibodies were as follows: 1:1,000 anti-cleaved Cas-3 (9664, Cell Signaling Technology, Danvers, MA, USA); 1:1,000 anti-CytoC (556433; BD, Franklin Lakes, NJ, USA); and 1:5,000 anti-COX (A21348; Thermo Fisher Scientific, Waltham, MA, USA). After incubation with secondary antibodies, protein bands were detected and captured, and the intensities were quantified with the Quantity One version 16.0 software (Bio-Rad Laboratories Inc., Hercules, CA, USA).