Fig. 5.

Genetic knock-in mutation of Ser74 of MNK2a to alanine in HEK293 cells enhances phosphorylation of eIF4E and mTORC1 downstream targets. a Cells were transfected with empty vector or the indicated GST-MNK constructs for 48 h, before lysates were made and subjected to immunoblot analysis. b Cells were serum starved for 16 h, pre-treated with 1 μM rapamycin or 1 μM AZD8055 before stimulation with 10 nM IGF-1 for 30 min. c MNK2 WT and S74A knock-in cells were treated with 1 μM rapamycin or 1 μM AZD8055 for 24 h before lysis and immunoblotting analysis was performed for the indicated P- or total proteins. d Lysates from MNK2 WT and S74A cells cultured in growth medium for 24 h were subjected to immunoblot analysis for the indicated proteins. e MNK2 WT and S74A cells were serum starved for 16 h, treated with rapamycin (1 μM) or AZD8055 (1 μM) for 30 min, and then stimulated with 10 nM IGF-1 for another 30 min. eIF4G, 4E-BP1 and eIF4E were isolated from lysates by m⁷GTP affinity chromatography and analysed by SDS-PAGE/WB against the indicated proteins from the eluates as well as the input lysates