A431 cells, plated and treated in parallel, were assessed for cell viability using an Alamar Blue assay. Data from A. Stattic or B. S31-201 treated cells are expressed as the average ± SEM (n=3) relative to cells incubated in 1.25% FBS in DMEM (reduced serum – RS). Serum-starved A431 cells were pre-treated for 1 hour with 0 (0.025% DMSO), 0.3, 1, 3, 5, or 10 μM Stattic (IC50 = 5.1 μM; panel A.), or with 0 (0.5% DMSO), 1, 3, 10, 30, and 100 μM S3I-201 (IC50 = 86 μM; panel C.), followed by the addition of 16 nM EGF (+) for 24 hours. Control cells were incubated with reduced serum media alone (RS), media containing 10% fetal bovine serum (FBS), or 16 nM EGF (EGF) for 24 hours. As a positive control cells were treated with 1 μM staurosporine (STS) for 4 hours. Cell lysates were prepared, 40 μg were resolved by SDS-PAGE, and immunoblotted for the indicated proteins. Fifteen μg of the STS sample were used to keep the immunoblot analysis in the dynamic range. Shown is a representative experimental replicate of three.