Pre-Treatment of Recombinant Mouse MFG-E8 Downregulates LPS-Induced TNF-α Production in Macrophages via STAT3-Mediated SOCS3 Activation
Figure 4
MFG-E8 induces SOCS3 expression in macrophages.
(A, B) RAW264.7 cells (4×106 cells) and (C, D) mouse peritoneal macrophages (2×106 cells) were cultured in a 6-well cell culture plates, and treated with rmMFG-E8 (500 ng/mL) for 2 h followed by assessment of SOCS3 mRNA expression and protein using real-time PCR and Western blot analysis, respectively. For real-time PCR, β-actin served as the internal control. To examine protein level of SOCS3, a total of 25 µg of proteins extracted from each samples were subjected for Western-blotting. The image shown is representative of results obtained from 3 independent experiments. The immunoblot was reprobed with mouse anti-β-actin Ab as loading control. Densitometric evaluations (SOCS3/β-actin) are expressed as means ± SE and compared by one-way ANOVA and SNK method: *P<0.05 vs. control. (E, F) RAW264.7 cells (4×106 cells) were cultured in a 6-well cell culture plate in presence of rmMFG-E8 (500 ng/mL) for 2 h, followed by LPS (10 ng/mL) stimulation for 2 and 4 h, respectively, for SOCS3 mRNA expression by real-time PCR and protein levels by Western-blotting. In each experiment, β-actin served as the internal control. Results are expressed as means ± SE and compared by one-way ANOVA and SNK method. *P<0.05 vs. control; #P<0.05 vs. LPS.