Caspase 3

Western blot analysis was performed as described previously (Binukumar et al., 2014 (link)). In brief, cells or brain tissues were lysed or homogenized in extraction buffer, respectively, and centrifuged at 10,000 × g for 5 min, and supernatants were collected and protein determined. Equal amounts of total protein (50 μg/lane) were resolved on a 4–20% SDS–polyacrylamide gel and blotted onto a polyvinylidene fluoride membrane. This membrane was incubated in blocking buffer/5% dry milk powder (wt/vol) for 1 h at room temperature, followed by incubation overnight at 4°C in primary antibodies. Primary antibodies used were as follows: Cdk5 (1:500), p35 (1:1000), TH (1:1000), cytochrome c (1:250) Bcl₂ (1:200), and Bax (1:200; all Biovision), β-actin (1:10,000; Sigma-Aldrich), caspase-3 (1:250), and tubulin (1:10,000; Sigma-Aldrich). The membranes were then washed four times in Tris-buffered saline/Tween-20 (5 min each), followed by incubation in respective secondary antibody (goat anti-mouse or goat anti-rabbit immunoglobulin (L)–horseradish peroxidase conjugate at a dilution of 1:3000) for 2 h at room temperature. Western blots were analyzed using the Amersham Biosciences ECL kit following the manufacturer’s instructions (GE Healthcare).

Chrysin (97% pure) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO), sodium selenite (Na₂SeO₃) was obtained from LOBA Chemie (Mumbai, India), and Dulbecco’s modified Eagle’s medium (DMEM) and the antibiotics streptomycin and penicillin were obtained from HiMedia (Mumbai, India). Antibodies for m-calpain, Lp82, EGR-1, COX-1, caspase-3, caspase-8, caspase-9, and β-actin were purchased from Sigma-Aldrich Chemical Co. and Cell Signaling Technologies (CST; Danvers, MA). Anti-rabbit immunoglobulin (IgG) secondary antibody was obtained from Genei (Bengaluru, India). All other chemicals and reagents were of analytical grade. Milli-Q water (Millipore, Bengaluru, India) was used throughout the experiments.