Fig 4. EGF and NGF promote ERK5 phosphorylation at Thr732 via ERK1/2 in HEK293 and PC12 cells.

(A) HEK293 cells were co-transfected with mycERK2 and mycERK5 wild-type (WT) or mycERK5 kinase-dead (KD) mutant. After the cells were preincubated in the presence or absence of U0126 (20 μM) for 30 min, they were stimulated with EGF (100 ng/ml) for 5 min. Then, ERK5 phosphorylation status at its TEY site and Thr732, phospho-ERK1/2 (TEY), mycERK5 and mycERK2 were examined by Western blotting. (B) PC12 cells were co-transfected with mycERK5 and mycERK2. After the cells were preincubated in the presence or absence of U0126 (20 μM) for 30 min, they were stimulated with NGF (100 ng/ml) or EGF (100 ng/ml) for 5 min. Then, ERK5 phosphorylation status at its TEY site and Thr732, phospho-ERK1/2 (TEY), mycERK5 and mycERK2 were examined by Western blotting.