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.