Figure 3. MFG-E8 activates pSTAT3.

Western-blot for pSTAT3 was done in extracted total proteins from (A) RAW264.7 cells (4×10⁶ cells) and (B) mouse peritoneal macrophages (2×10⁶ cells) grown in 6-well plate and treated with rmMFG-E8 (500 ng/mL) for 2 h. (C) RAW264.7 cells were cultured and then stimulated with LPS (10 ng/mL) for 2 h. Western-blot analysis was performed from 25 µg of extracted proteins from the above described experiments using anti-mouse pSTAT3 Ab. The immunoblots were stripped and reprobed with anti-mouse STAT3 Ab. The images shown are representatives of 3 independent experiments. Densitometric evaluations (pSTAT3/STAT3) are expressed as means ± SE and compared by one-way ANOVA and SNK method: *P<0.05 vs. control. (D) Total protein was extracted from RAW264.7 cells (1×10⁶ cells) grown in 24-well culture plates and treated with rmMFG-E8 (500 ng/mL) and LPS (10 ng/mL) at different time points, and Western-blot for pSTAT3 and STAT3 was performed as described above. A representative autoradiograph is also shown. Densitometric evaluations (pSTAT3/STAT3) are expressed as means (n = 3) ± SE and compared by one-way ANOVA and SNK method: *P<0.05 vs. LPS at 0 h; ^# P<0.05 vs. LPS at corresponding time points. (E, F) RAW264.7 cells (1×10⁶ cells) were cultured in a 24-well cell culture plate and treated with Static (6.25 µM) for overnight, followed by pre-treatment with rmMFG-E8 (500 ng/mL) for 2 h and stimulated with LPS (10 ng/mL) for another 4 h. TNF-α mRNA and protein were measured by real-time PCR and ELISA, respectively. For real-time PCR, β-actin served as the internal control. Data are expressed as means (n = 3) ± SE and compared by one-way ANOVA and SNK method: *P<0.05 vs. LPS (−); ^# P<0.05 vs. LPS (+).