FIG 5.

TTP regulates CTSB mRNA. (A) AREsite (http://rna.tbi.univie.ac.at/cgi-bin/AREsite.cgi) was used to screen the 3′ UTR of CTSB for AU-rich elements, and one site was predicted for TTP binding. (B) Primary human macrophages were transfected with siRNA against TTP (siTTP). The knockdown was validated by analyzing mRNA levels of TTP normalized to TBP and compared to the scrambled control. (C) Macrophages were transfected with siRNA (50 nM) against TTP (siTTP) or a scrambled control siRNA (scr) and incubated for 8 h under hypoxia. CTSB mRNA was measured using qPCR. (D) Primary human macrophages were time-dependently exposed to hypoxia. TTP, phospho-TTP (pTTP), and nucleolin were analyzed by Western blotting. Different exposure times were used to obtain appropriate pTTP or TTP signals. (E) Macrophages with a knockdown of TTP were incubated for 8 h in hypoxia, with ActD (2.5 μg/ml) added for the last 4 h. mRNA of CTSB was measured and normalized to 18S rRNA (black bars). Macrophages were incubated for 8 or 48 h under hypoxia, with ActD added for the last 4 h. These graphs (white bars) were created from data shown in Fig. 4C. Data are expressed as means ± SEM (n ≥ 3 experiments). *, P < 0.05.