FIG 6.

JNK influences CTSB mRNA expression and stability. Primary human macrophages were incubated with DMSO, LY294002 (10 μM), SB203580 (10 μM), or SP600125 (20 μM) under hypoxia for 8 or 48 h. (A) CTSB mRNA was analyzed using qPCR. (B) Macrophages were time-dependently exposed to hypoxia, and protein amount of c-Jun N-terminal kinase (JNK) and phosphorylated JNK was measured by Western analysis, while nucleolin served as a loading control. (C) Macrophages were incubated for 0 to 48 h under hypoxic conditions, and JNK mRNA was analyzed by qPCR. Data were normalized against TBP. (D) Primary human macrophages were incubated for the indicated time periods under hypoxia and treated with either SP600125 (20 μM) or DMSO. Western analysis was performed using an antibody against TTP or nucleolin (loading control). Blots were exposed to films for variable times to obtain appropriate pTTP or TTP signals. (E) Macrophages were incubated with SP600125 (20 μM) under hypoxia for 48 h, and transcription was blocked by adding actinomycin D (ActD; 2.5 μg/ml) during the last 4 h. CTSB mRNA was measured and normalized to 18S rRNA (0-h ActD control = 1). Data are expressed as means ± SEM (n ≥ 3 experiments). *, P < 0.05.