p38 positively regulates c-Jun degradation. (A, Left panel) RAW264.7 cells were transfected with siRNA against p38. The knockdown level of p38 was measured through real-time PCR. (A, Middle panel) Two days after transfection, the cells were treated with LPS (1 μg/ml) for the indicated times. Immunoblotting analysis was performed in order to detect the levels of p38 and c-Jun among RAW264.7 cells with p38-knockdown after LPS stimulation. (A, Right panel) Immunoblotting analysis was carried out in order to detect the levels of p38 and c-Jun among RAW264.7 cells with p38-knockdown during either SO or LPS exposure. (B) RAW264.7 cells were transfected with Flag-p38 for the indicated times, and then Flag and c-Jun levels were analyzed through immunoblotting analysis. (C) HEK293T cells were transfected with Flag-p38 and Myc-c-Jun for 24 hours, followed by SO treatment. After preparing cell lysates, immunoprecipitation and immunoblotting analyses were performed in order to check the levels of Flag and Myc. (D) IP-kinase assay was then performed in the presence or absence of enzyme (immunoprecipitated p-p38) and substrate (immunoprecipitated c-Jun). Immunoblotting analysis was then performed to determine the levels of phospho-c-Jun and c-Jun. (E) HEK293T (2 × 106 cells/ml) cells were then transfected with HA-p38 and CFP-TRIF for 24 hours. The levels of p-p38 and c-Jun were detected through immunoblotting analysis. (F) RAW264.7 cells were treated with Anisomycin (Aniso) for 24 hours at the indicated concentrations. The levels of p-p38 and c-Jun were detected through immunoblotting analysis. #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 Middle panel) si-Con without LPS (0 min), (A Right panel) without LPS treatment, (C) Flag-p38 (−) + Myc-c-Jun (−) + SO (−), (E) HA-p38 (−) + CFP-TRIF (−), (F) Aniso (−). Control group: (A Left panel) si-Con, (A Right panel) si-Con + LPS, (B) Flag-p38 (−), (C) Myc-c-Jun (+), (D) ATP (+) + IPed-c-Jun (+), IPed-p-p38 (−), (E) CFP-TRIF (+). Blue notation: (A Middle panel) compared to the same time points, (A Right panel) compared with si-Con + SO (+).
