Mouse anti nitrotyrosine

The Western blotting was conducted as described earlier (Huang et al., 2018 (link)). Briefly, the ischemic cortex was collected at different time after reperfusion, homogenized in tissue lysing buffer. Proteins were separated with 8%–12% SDS-polyacrylamide gel and transferred to nitrification cellulose membranes with glycine transfer buffer. The membrane was sealed with 5% skim milk and protein was detected with primary antibody, including mouse anti-nitrotyrosine (#05233, Millipore, Darmstadt, Germany), mouse anti-γH2. AX (#ab2893, Abcam, Cambridge, United Kingdom), rabbit anti-4-hydroxynonenal (#ab46545, Abcam, Cambridge, United Kingdom), anti-SIRT3 (#2627, Cell Signaling Technology, Danvers, United States), anti-PGC-1α antibody (#SC13067, Santa Cruz, Dallas, United States), and mouse anti-GAPDH (#ab9484, Millipore, Darmstadt, Germany), anti-β-Actin (#A5441, Sigma, Darmstadt, Germany). After incubating overnight at 4°C, the membrane was incubated with secondary antibodies including anti-mouse IgG or anti-rabbit IgG (Li-Cor Bioscience, Lincoln, NE, United States). The protein bands were visualized using the Odyssey Infrared Imaging System (Li-Cor Bioscience). After the images were collected, the protein expression was finally determined using ImageJ Launcher software (National Institutes of Health, Bethesda, MD, United States) and normalized to a loading control GAPDH.