HEK 293 cells were serum starved for 8 hours and then incubated for 20 mins without (-) or with (+) 100 nM wortmannin before stimulation for a further 15 mins with 20 ng/ml IGF-1 or for 30 mins with 400 ng/ml PMA. The cells were lysed and METTL1 was immunoprecipitated from 1.5 mg of extract using 5 μg anti-METTL1 (S177B). The immunoprecipitate was denatured in SDS, subjected to SDS/PAGE and transferred to nitrocellulose membranes. The membranes were immunoblotted with an antibody that recognises METTL1 phosphorylated at Ser 27 and with an antibody that recognises the phosphorylated and unphosphorylated forms of METTL1 equally well (anti-METTL1 S177B). Binding of the primary antibody was detected using rabbit peroxidase conjugated anti- sheep IgG antibody (1 in 10, 000 dilution, Pierce) followed by enhanced chemiluminescence (ECL, Amersham).