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 (anti-METTL1 phospho Ser 27 S793A) 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).