Af5128

Rats were dissected on ice, and tissue was homogenized in lysis buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1% SDS P40, 5 mM EDTA, and protease inhibitors (Complete Mini; Roche). Cellular debris was removed by centrifugation at 14,000 rpm for 20 min at 4°C, and the supernatant was collected for analysis. Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a 5% stacking gel and a 10% separation gel to separate 20 L of protein in running buffer. The stacking gel was run at 80 V for 0.5 h, and the separation gel was run at 100 V for 1.5 h. The separated proteins were then electrotransferred onto a nitrocellulose filter membrane in transfer buffer at 180 mA for 1.5 h. Protein blots were blocked with 5% defatted milk for 30 min and probed with specific antibodies against TNF-α (Abcam, ab62609), IL-1ꞵ (Affinity, AF5103), Beclin-1 (Affinity, AF5128), and cleaved-caspase3 (Affinity, AF7022). Anti-α-tubulin antibody (Sigma, T6557) was used as a loading control. After three washes with TBST, secondary antibody (CWS) was added at room temperature for 1 h using 5% milk in TBST followed by three additional washes with TBST. Bands were visualized using the Immobilon Western ECL system and were analyzed with the software Gel Pro Analysis (24).