Anti cleaved caspase 3

Cells were harvested and lysed in RIPA lysis buffer (Beyotime, Shanghai, China) that included a protease inhibitor cocktail (19 (link)) (Sigma-Aldrich; Merck KGaA). Protein quantification was performed using the Bio-Rad Protein assay dye reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins were electrophoresed and separated by SDS-PAGE, and then transferred from gels onto PVDF membranes (EMD Millipore, Billerica, MA, USA). After incubation with primary (4°C, overnight) and secondary antibodies (room temperature, 2 h), the blots were developed with DAB (3,3′-diaminobenzidine). Western blots results were analyzed with GIS 1D gel image system software. The following primary antibodies were used: anti-CLCN3 (1:100, sc-17572), anti-Bcl-2 (1:200, sc-783), anti-Bax (1:200, sc-7480), anti-pro-caspase 3 (1:200, sc-7148), anti-cleaved-caspase 3 (1:200, sc-22171), anti-cathepsin D (1:200, sc-136282), anti-β-actin (1:400, sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Secondary HRP-conjugated antibodies were purchased from Thermo Fisher Scientific, Inc. Densitometry was used to calculate the ratio of CLCN3 to β-actin, cleaved caspase 3 to pro-caspase 3, and BCL2 to BAX signal in each lane.

Bicinchoninic acid (BCA), trichloroacetic acid (TCA), sulforhodamine B (SRB), propidium iodide, RNase A, dimethyl sulfoxide (DMSO) and catalase were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco Modified Eagle Medium (DMEM)/high glucose, Minimum Essential Media (MEM), fetal bovine serum (FBS), trypsin-EDTA solution (1X), antibiotic-antimycotic solution (100X), and PBS (1X) were purchased from HyClone Laboratories, Inc. (South Logan, UT, USA). Anti-caspase-9, anti-cleaved caspase-9, anti-caspase-3, anti-BID, anti-p-p53 (Ser15), anti-Prx1, anti-caspase-8, and anti-cleaved caspase-8 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-cleaved caspase-3, anti-Bax, anti-Bcl-2, anti-p53, anti-catalase, anti-SOD-1, anti-SOD-2, anti-GPx-1, anti-Trx, anti-PrxI/II, anti-β-actin, and all secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-BIM, anti-PARP, anti-cleaved PARP, and fluorescein isothiocyanate (FITC) Annexin V apoptosis detection kit I were purchased from BD Biosciences (CA, USA). Anti-RIP-3 was purchased from Abcam (Cambridge, MA, USA). 2′,7′-dichlorodihydrofluorescein diacetate (DCFH₂-DA) was purchased from Invitrogen (Carlsbad, CA, USA).

Twenty-four h after cerebral ischemia-reperfusion, the appropriate amount of brain tissue from each group was placed in a cell lysate (Beyotime, China) to extract total protein. The protein concentration was determined using a bicinchoninic acid (BCA) assay kit (Beyotime, China). Next, a 5× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer was mixed with the sample protein and heated in a 95–97 °C water bath for 5 min, and then stored in a −80 °C refrigerator for the subsequent experiment. The quantified amount of protein was loaded and separated by 12% PAGE, and a polyvinylidine difluoride (PVDF) membrane was used to transfer the protein. 5% skim milk was used to block at room temperature for 1 h, and anti-phosphorylated (p)-p38MAPK (1:3,000, Cell Signaling Technology, USA), anti-cleaved caspase-3 (1:2,000, Santa Cruz Biotechnology, USA), and β-actin (1:1,000, Santa Cruz Biotechnology, USA) were added and incubated overnight at 4 °C. The second antibody was added and incubated for 2 h at room temperature the following day. After washing the membrane with tris-buffered saline, 0.1% Tween 20 (TBST), Image Lab 3.0 was used for imaging. ImageJ software was used to quantitatively analyze the difference in protein expression in each group. β-actin was used as an internal reference for the quantitative measurement of protein expression.