Inhibition of RAS-GTP and phosphorylated ERK1/2 in human MPNST cells exposed to Sulindac derivatives A: Cell lines S462 and S520 were grown in DMEM with 10% serum and treated at 80–90% confluency with either 0.2% DMSO, 500 μM Exisulind (Exi) or 125 μM Sulindac Sulfide (s. s.). Cells were lysed after 24 h and RAS was immunoprecipitated and detected by western blotting with an anti-RAS antibody, the same lysates were blotted for phosphorylated (phospho-) and basal (total-) ERK1/2. B: Upregulation of phosphorylated ERK1/2 and AKT for treated and untreated cells after addition of EGF, whereas the RAS-GTP level remains unchanged at all conditions. Cell line S462 was starved over night and then Sulindac metabolites were supplemented at concentrations mentioned above in DMEM containing 0.1% serum. EGF was added after 24 h of treatment, cells were lysed 15 min later and after western blotting incubated with phosphorylated and unphosphorylated ERK1/2, phospho-AKT and RAS-antibodies.