Horseradish peroxidase conjugated goat anti rabbit antibody

After 24 h of reperfusion, the mice (n = 6, for each group) were decapitated and ischemic brains were collected. Protein lysates of ischemic hemispheres in each group were subjected to 8% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was treated with blocking solution (5% skim milk in TBST) and incubated overnight at 4°C with the primary rabbit monoclonal antibodies respectively (Bax, 1:1000; Bcl-2, 1:500; Caspase-3, 1:1000; Caspase-9, 1:500; Proteintech Group, U.S.A.; CytC, 1:1000; Abcam, U.K. and PARP-1, 1:500; Cell Signal, U.S.A.). Secondary incubations were performed with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:3000; Proteintech Group, U.S.A.). An anti-actin antibody (1:1000; Proteintech Group, U.S.A.) was used as loading controls. Immunoreactive proteins were visualized using enhanced chemiluminescence (ECL) and the signals were quantified by densitometry with a Western blotting detection system (Quantity One, Bio-Rad, U.S.A.).

Lipopolysaccharide (from Escherichia coli, 055:B5) and 2-DG were the products of Sigma (St. Louis, MO, USA). The PKM2 activator ML265, the STAT3 inhibitor stattic, and the myeloperoxidase (MPO) activity assay kit were the products of Cayman Chemical (Ann Arbor, MI, USA). The enzyme-linked immunosorbent assay (ELISA) kits for the determination of mouse tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) were the products of NeoBioscience Technology Company (Shenzhen, China). The nuclear protein extract kit was the product of Genechem Co., Ltd. (Shanghai, China). The rabbit anti-mouse PKM2 (D78A4), STAT3 (79D7), phospho-STAT3 (D3A7), and lamin B (D9V6H) antibodies were the products of Cell Signaling Technology (Danvers, MA, USA). The horseradish peroxidase-conjugated goat anti-rabbit antibody was the product of Proteintech (Wuhan, China). The BCA protein assay kit was the product of Thermoscientific (Rockford, IL, USA). The enhanced chemiluminescence (ECL) reagents were the products of Advansta (Menlo Park, CA, USA).