p38 MAPK is involved in the transcriptional regulation of IL12RB1. (A) RAW264.7 cells were treated with 10 ng/mL IFN-γ, 10 ng/mL IL-15, or 1 μg/mL LPS for the indicated time before total cellular lysate preparation. The phosphorylation of p38 (pp38) or JNK (pp54 and pp46) was detected by Western blot analysis using polyclonal antibody (pAb) specific for the phosphorylated p38 or mAb specific for the phosphorylated JNK. As a control, 10% of each lysate was used to detect p38 or JNK using anti-p38 or anti-JNK1 pAb, respectively. (B) RAW264.7 cells stably integrated with pGL3-2508 luciferase construct were prepared as described for Figure 2B. Cells were left untreated or were pretreated with 50 μM SB203580 or 50μM SP600125 for 30 minutes before stimulation with 10 ng/mL IFN-γ or 10 ng/mL IL-15 for an additional 8 hours. Lysates were prepared for luciferase assay. Basal luciferase activity of untreated cells was generally similar among all the examined clones, and the fold inductions are expressed as described for Figure 2B. (C) RAW264.7 cells were pretreated with SB203580 for 30 minutes before stimulation with 10 ng/mL IFN-γ or 10 ng/mL IL-15 for the indicated time. The acetylation of histone H3 was analyzed as described for Figure 4E. (D) RAW264.7 cells were treated as described in panel C. Nuclear lysates were prepared as previously described.32 The phosphorylation of histone H3 (p-H3) was detected by Western blot analyses using an antibody specific for the phosphorylated histone H3. As a control, 10% of each lysate was used to measure protein levels by Coomassie staining.
