Odyssey clx imaging machine

RAW 264.7 macrophage was pretreated with 5-hydroxymaltol for 1 h and stimulated with LPS (1 µg/mL) for 30 min. The macrophage cells were harvested and lysed using a radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Waltham, MA, USA). Each sample was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electro-transferred to Immobilin-FL polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA). After blocking with Odyssey^® Blocking Buffer (LI-COR, Lincoln, NE, USA) for 1 h, the blot was incubated overnight with the primary antibodies at 4 °C. After being washed, the blots were incubated with diluted IRDye 680- and IRDye 800-labeled secondary antibodies using Odyssey blocking buffer containing 0.2% Tween-20/0.01% sodium dodecyl sulfate for 60 min. The protein bands were detected with an Odyssey CLx imaging machine (LI-COR). The Primary antibodies against pp38, JNK, pJNK, ERK, pERK, p65, Nrf2 and HO-1 were obtained from Cell Signaling Technology (Beverly, MA, USA). The antibodies of p38 and β-actin were purchased from Santa Cruz Biotechnology, Inc (Dallas, TX, USA).