HEK 293 cells were derived of serum for 15 hours then stimulated for 1 hour without (-) or with (+) 2 μM PD 184352 or for 15 mins without (-) or with (+) 100 nM wortmannin, before being stimulated for 5 or 30 mins with 20 ng/ml of IGF1. The cells were then lysed and the lysates (45 μg of protein) were subjected to SDS-PAGE, then transferred on to nitrocellulose membrane. The membranes were then probed with antibodies that recognise CRHSP24 phosphorylated at Serine 52 (anti-CRHSP24 phospho Ser 52) and that recognise unphosphorylated and phosphorylated CRHSP24 equally as well. 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).