Figure 3
Inhibitors of TLR4 and PI3K suppress GOS-induced activation of Akt/NF-κB and Akt/mTOR pathways. (A) RAW264.7 cells were pre-incubated with the TLR4 inhibitor TAK-242 (1 μM) or PI3K inhibitor LY 294002 (10 μM) for 2 h and then co-treated with 1 mg/ml GOS for the indicated time. Akt phosphorylation was analyzed by Western blotting. (B) p-IκB, IκB and NF-κB p65 in the cytosolic fraction and p65 in the nuclear fraction were detected by Western blotting. Levels of phosphorylated and nonphosphorylated mTOR and p70 S6K were analyzed by Western blotting. (C) RAW264.7 cells were either pre-treated with the TLR4 inhibitor TAK-242 (1 μM), PI3K inhibitor LY 294002 (10 μM) or mTOR inhibitor rapamycin (50 μM) for 2 h prior to GOS treatment or treated with inhibitors alone. iNOS protein expression was detected by Western blotting. The full-length western blots were presented in supplementary information. (D) Nitrite production in the culture supernatant of treated cells was measured by the Griess assay. (E) TNF-α in the culture supernatant from treated cells was measured by ELISA. LPS (1 μg/ml) was used as a positive control. Representative images and results from three independent experiments are shown. #Indicates significant differences between the control and GOS-treated groups, ### P < 0.001. *Indicates significant differences between the GOS-treated and GOS with inhibitor-treated groups, **P < 0.01, ***P < 0.001.
