Fig. 3

EGF regulates NTN4 to stimulate the phosphorylation of AKT, ERK, and Src. U251MG cells were treated with EGF (40 ng/mL) and NTN4 (50 ng/ml) for 48 h. Then these cells were treated with NTN4 (50 ng/mL) or EGF (40 ng/mL) for 10 min or 30 min. Subsequently, cells were lysed and the phosphorylation of AKT, ERK, Src and mTOR detected. a Short term stimulation of EGF significantly enhanced AKT phosphorylation, and a similar phenomenon was observed after 48 h of stimulation with recombinant NTN4. Addition of exogenous NTN4 increased p-AKT levels at 10 min and 30 min. After EGF stimulation for 48 h, NTN4 was not able to further increase p-AKT. Long term addition of exogenous NTN4 did not affect p-ERK. However, short term stimulation of EGF significantly enhanced ERK phosphorylation, regardless of long term NTN4 stimulation. Addition of exogenous NTN4 increased levels of p-ERK at 30 min. p-ERK did not show any significant change after 48 h of stimulation with EGF. b Long term addition of exogenous NTN4 did not affect p-Src. Stimulation with EGF for 10 min significantly increased p-Src, with or without 48 h of stimulation with exogenous NTN4. Stimulation with EGF for 30 min enhanced p-Src, which was abolished after treatment with NTN4 for 48 h. Addition of exogenous NTN4 increased p-ERK after 10 min. Although p-Src was strongly up-regulated after 48 h of treatment with EGF, NTN4 did not additionally stimulate Src phosphorylation. Long term addition of exogenous NTN4 did not affect p-mTOR. EGF significantly increased p-mTOR levels at both 10 min and 30 min, with or without 48 h stimulation with exogenous NTN4. Addition of exogenous NTN4 significantly enhanced p-mTOR at 30 min. EGF treatment for 48 h did not change p-mTOR levels; however, p-mTOR levels were strongly increased when combined with NTN4 stimulation for 30 min. Mean ± SE, n ≥ 3, * p-value < 0.05, ** p-value < 0.01