Anti bcl 2 50e3

To quantify protein expression levels, brain slices were collected either after OGD or OD treatment after Il4rα-specific siRNA transfection. Apoptosis was determined by measuring apoptotic factor c-Caspase 3 and anti-apoptotic factor BCL-2. Brain slices were homogenized in cold lysis buffer (50 mM Tris-HCl [pH 7.8], 150 mM NaCl, 0.2% Triton X-100) containing protease and phosphatase inhibitor cocktail (Thermo Scientific). Protein samples (50–100 µg) were electrophoresed on a 10% to 15% polyacrylamide gel and then transferred on PVDF membrane for 90 min on ice. Membranes were incubated with primary and secondary antibodies, and the level of protein was visualized via chemiluminescence (ECL Detection Kit, Thermo Fisher Scientific). The following primary antibodies were used: anti-IL-4Rα (H-4) (1:1,500, Santa Cruz Biotechnology Inc., sc-28361), anti–phospho-STAT6 (Tyr641) (1:1,500, BD Pharmingen, 558241), anti-STAT6 (M-20) (1:2,000, Santa Cruz Biotechnology Inc., sc-981), anti-cleaved Caspase 3 (Asp175) (1:1,500, Cell Signaling Technology, 9661), anti-BCL-2 (50E3) (1:2,000, Cell Signaling Technology, 2870), and anti-GAPDH (1:4,000, Santa Cruz Biotechnology, sc-32233). HRP-conjugated anti-mouse and anti-rabbit (Santa Cruz Biotechnology Inc.) secondary antibodies were used to detect proteins.