Figure 4
From: Central role of myeloid MCPIP1 in protecting against LPS-induced inflammation and lung injury
Pharmacological inhibition of MALT1 with MI-2 selectively increased the abundance of the MCPIP1 protein and suppressed LPS-induced macrophage activation. (a, b) Raw264.7 cells were treated with MI-2 for different durations or doses as indicated. Cell lysates were subjected to analysis by immunoblot with the MCPIP1 antibody. β-actin served as a loading control. Fold changes of the MCPIP1 protein levels were determined by densitometry and normalized to β-actin. Data are representative of three independent experiments. (c) Raw264.7 were pretreated with or without 1 μM MI-2 for 1 h and then stimulated with or without 1 μg/ml LPS for 4 h. The protein levels from different genes were determined by Western blot with individual antibodies as indicated. β-actin served as a loading control. (d) Fold-change of the MCPIP1 and C/EBPδ protein levels were determined by densitometry and normalized to β-actin. Data are presented as the means±s.d. (n=3), *P<0.05 by Student’s t-test. (e) Raw264.7 were pretreated with or without 1 μM of MI-2 for 30 min and then stimulated with or without 1 μg/ml LPS for 4 h. The mRNA levels of selected cytokines were determined by qPCR analysis and normalized to the β-actin mRNA levels. Data are presented as the means±s.d. (n=4), *P<0.05 by Student’s t-test. (f) Raw264.7 cells were transfected with si-Control or si-MCPIP1 for 24 h. The transfected cells were pretreated with or without 1 μM MI-2 for 1 h and then stimulated with or without 1 μg/ml of LPS for 4 h. The protein levels of different genes were determined by immunoblot with specific antibodies. β-actin served as a loading control. (g) Fold-change of the MCPIP1 and C/EBPδ protein levels were determined by densitometry and normalized to β-actin. Data are presented as the means±s.d. (n=3), *P<0.05 by Student’s t-test. (h) Raw264.7 cells were transfected with si-Control or si-MCPIP1 for 24 h. Transfected cells were pretreated with or without 1 μM MI-2 for 1 h and then stimulated with or without 1 μg/ml LPS for 4 h. The mRNA levels of MCPIP1 and selected cytokines were determined by qPCR analysis and normalized to actin mRNA levels. Data are presented as the means±s.d. (n=4), *P<0.05 by Student’s t-test.
