Cleaved caspase 3

At reperfusion 24 hr after MCAO injury, rat were sacrificed and brains were washed rapidly with ice-cold PBS, and collected. Tissues were lysed with ice-cold RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA). The lysates were centrifuged at 13,200 rpm for 1 hr at 4℃ to produce whole-cell extracts. Protein content was quantified using the BCA method (Pierce, Rockford, IL, USA). Protein (20 µg) was separated on a 10% SDS-polyacrylamide (PAGE) gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% bovine serum albumin, prepared in Tris-buffered saline/Tween (TBS-T; 20 nM Tris [pH 7.2], 150 mM NaCl, and 0.1% Tween 20), for 1hr at room temperature (RT), immunoblots were incubated overnight at 4℃ with primary antibodies that specifically detect Bcl 2 associated X protein (Bax) (1:1000, Abcam, Cambridge, MA, USA), Cleaved caspase-3 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), or β-actin (1:2000, Cell Signaling Technology, Danvers, MA, USA). Next, blots were incubated with HRP-linked anti-mouse and -rabbit IgG antibodies purchased from Abcam (Cambridge, UK) for 1 hr at RT. Enhanced chemiluminescence was performed by ECL (Pierce) [34 (link)].

Total cellular protein was extracted from A549 cells using RIPA lysis buffer (Beyotime, Shanghai, P.R. China). Proteins were quantified using a BCA Protein Assay Kit (Pierce, Bonn, Germany). Then samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Whatman, Dassel, Germany). The blots were blocked in 5% skim milk at room temperature for 1 h and incubated overnight with specific primary antibodies: Bax, Bcl-2, cleaved caspase 3, pro caspase 3, CDK2, cyclin E, p27, p21, c-Jun, p-c-Jun, p65, p-p65, IκB-α, p-IκB-α, BTG2, p53, and GAPDH (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C. Afterward, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 2 h. The bands were imaged using the WEST-ZOL-plus Western Blot Detection System.