Figure 3

Identification of C/EBPβ and C/EBPδ mRNAs as the targets of MCPIP1 RNase. (a) qPCR analysis of the mRNA levels for MCPIP1, C/EBPβ, C/EBPδ, and other inflammatory cytokines in BMDMs from Mcpip1^flfl and M-Mcpip1^−/− mice treated with LPS for the indicated times. Data are representative of three independent experiments with 3 mice per group. *P<0.05 by Student’s t-test. (b) Immunoblot analysis of the C/EBPβ and C/EBPδ protein levels in the lungs of Mcpip1^flfl and M-Mcpip1^−/− mice treated with PBS or LPS for 8 h. β-actin served as a loading control. (c) Fold-change of C/EBPβ and C/EBPδ proteins in experiments presented in (b) were determined by densitometry and normalized to β-actin. **P<0.01, *P<0.05 by Student’s t-test. Data are representative of three independent experiments. (d) BMDMs from Mcpip1^flfl and M-Mcpip1^−/− mice were stimulated with LPS (1 μg/ml) for 4 h and then incubated with AcD (5 μg/ml) for different times as indicated. The mRNA levels were measured by qPCR and normalized to the actin mRNA levels. Data were presented as the means±s.d., n=4. (e) The reporter of Luc-Control or Luc.-mouse C/EBPδ 3′UTR or Luc.-human C/EBPδ 3′UTR was transfected with or without Flag-MCPIP1 into Raw264.7 cells. Twenty-four hours later, cells were collected for analysis of luciferase activity and normalized to control renilla activity. N=4, *P<0.05. **P<0.01 versus vector. Data are representative of three independent experiments. (f) The stem-loop structures on human C/EBPδ 3′UTR identified by mfold. (g) The serial deletion reporters, as indicated, were transfected with or without Flag-MCPIP1 into Raw264.7 cells. Twenty-four hours later, cells were collected for analysis of luciferase activity and normalized to control renilla activity. N=4, **P<0.01 versus vector. Data are representative of three independent experiments.