Figure 5

mTOR regulates eosinophil differentiation via p-Erk signalling. (a) Blots for p-Erk and Erk were displayed from the same gel. Phospho-Erk was firstly detected, then the antibody was eluted by 200 mM sodium hydroxide solution, and total Erk antibody incubation followed using the same membrane. Other blots were cropped by different gels.) Erk signal and p-Erk were detected using western blot on day 9. (b–e) On day 9, cells were harvested using Trizol reagent and mRNA performed inverse transcription. Quantitate PCR was performed to detect major basic protein (Mbp) and Gata-1 expression. Result B showed Mbp expression after adenoviral administration and result C displayed Gata-1 expression after adenoviral exposure. Result D revealed Mbp expression with torin-1 (250 nM) treatment and result E suggested Gata-1 expression after torin-1 exposure. (f,g) U0126 (20 μM) was used to inhibit phosphorylation of Erk, treatment of U0126 resulted in diminished eosinophil development in both strains (f). Results were analysed as means ± SEM (g). (h,i) Mbp (h) and Gata-1 (i) were also detected by Q-PCR post U0126 administration. Results were analysed as means ± SEM. All the results above were repeated by three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.