Figure 4.

Effects of IGF1 and PMA on the phosphorylation of endogenous METTL1 in HEK293 cells. (A) Cells were serum starved for 8 h and then incubated for 20 min without (−) or with (+) 100 nM wortmannin before stimulation for a further 15 min with 20 ng/ml IGF-1 or for 30 min with 400 ng/ml PMA. METTL1 was immunoprecipitated from 1.5 mg cell lysate protein, denatured in SDS, subjected to SDS–PAGE and, after transfer to Immobilon-P membranes, immunoblotted with the antibody that recognises METTL1 phosphorylated at Ser27 and with the antibody that recognises phosphorylated and unphosphorylated METTL1. A further 20 μg of lysate protein was subjected to SDS–PAGE and immunoblotted with an antibody that recognises PKB phosphorylated at Thr308, an antibody that recognises phosphorylated and unphosphorylated PKBα equally well, an antibody that recognises ERK1 and ERK2 phosphorylated at the Thr-Glu-Tyr motif (pTEpY) and an antibody that recognises all forms of the ERK1 and ERK2 proteins. (B) Bacterially expressed human GST-METTL1 was left unphosphorylated (no kinase, NK) or maximally phosphorylated with PKBα or RSK2 and 100 ng aliquots spotted onto a nitrocellulose membrane and immunoblotted with the antibody that recognises METTL1 phosphorylated at Ser27 and with the antibody that recognises unphosphorylated and phosphorylated METTL1 equally well. (C) Same as panel A, except that after serum starvation for 8 h, the cells were incubated for 1 h in the absence or presence of 2 μM PD 184352 (instead of wortmannin) prior to stimulation with IGF-1 or PMA. (D) Same as panel A, except that following serum starvation the cells were incubated for 1 h in the absence or presence of 100 nM rapamycin (rather than wortmannin) prior to stimulation with IGF-1 or PMA. In addition, 20 μg cell lysate was immunoblotted with an antibody that recognises S6K1 phosphorylated at Thr389 and with an antibody that recognises phosphorylated and unphosphorylated S6K1 equally well.