Western Blot
RAW264.7 cell were transfected by siRNA or plasmid, hydrolyzed in RIPA lysis buffer for 30 minutes after stimulated with LPS for 8 hours. The BCA protein were employed to detect the protein concentration, then separated by 10% SDS-PAGE. Membranes were blocked in 5% milk for 1 hour and then treated with antibodies overnight. The primary antibodies were: anti-PED4B antibody, anti-cAMP antibody, anti-NR4a1 antibody were bought from Abcam Technology, USA. Anti-PKA antibody, anti-phosphorylated-PKA antibody, anti-p65 antibody, anti-phosphorylated-65 antibody, anti-IκBα antibody, anti-phosphorylated antibody, anti-GAPDH antibody were purchased from Cell Signaling Technology, USA. After washed and incubated with mouse or rabbits secondary antibodies for 1 hour, the protein bands were detected using the enhanced chemiluminescence reagents.