20 μg of unstimulated (U) and IGF stimulated (S) 293 cell lysates were separated by SDS-Page transferred to nitrocellulose membrane and blotted with the anti-ribosomal S6 phospho Ser 235 antibody at 1 μg/ml in the presence of 10 μg/ml of the non- phosphorylated peptide. Binding of the primary antibody was detected using rabbit peroxidase conjugated anti-sheep IgG (1 in 10, 000 dilution, Pierce) followed by enhanced chemiluminscence (ECL, Amersham).