p38-mediated c-Jun degradation is ubiquitin-dependent. (A) RAW264.7 cells (1 × 106 cells/ml) were pre-treated with SO (400 μM), followed by LPS (1 μg/ml) incubation for 45 minutes, and then further treated with MG132 (30 μM) 4 hours before harvesting the cells. The levels of c-Jun were analyzed by immunoblotting analysis. (B) HEK293T cells were transfected with HA-p38 and CFP-TRIF for 24 hours. MG132 (15 μM) was then added 4 hours prior to cell harvesting. The protein levels of p-p38, c-Jun, and β-actin and mRNA levels of c-Jun and GADPH were then analyzed through immunoblotting and RT-PCR. (C) HEK293T cells were transfected with HA-p38 and CFP-TRIF for 24 hours. MG132 (15 μM) was then added 4 hours prior to cell harvesting. Cell lysates were then subjected to immunoprecipitation with c-Jun antibodies, and the levels of Ub were detected through immunoblotting analysis. (D) A schematic illustration depicting the dominant role of p38 in the regulation of c-Jun degradation. #P < 0.05 or ##P < 0.01 compared to the normal group and *P < 0.05 or **P < 0.01 compared to control groups. Normal group: (A) without treatment, (B; C) MG132 (−) + HA-p38 (−) + CFP-TRIF (−). Control group: (A) LPS alone, (B; C) HA-p38 (+) + CFP-TRIF (+).
