Figure 1. Ctx^R clones have increased AKT signaling pathway. (A) Ctx^R clones are dependent on AKT. Ctx^R clones were plated and treated with 50 nM of AKT1/2(a) siRNA, 50 nM of AKT1/2(b) siRNA or 50 nM non-targeting siRNA. Cell proliferation was measured at 96 h after treatment using the proliferation assay described in materials and methods. Data points are represented as mean ± SEM (n = 4). *p ≤ 0.05. Protein was collected at 96 h after treatment and fractioned by SDS-PAGE and immunoblotted for AKT and phopho-rpS6. α-Tubulin was used as a loading control. (B) Fold increase in expression of phosphorylated proteins in Ctx^R HC4 cells compared with parental control HP cells by phosphoprotein arrays. Ctx^S parental cells (HP) and Ctx^R clones (HC4) were harvested and lysed with the extraction buffer provided as described according to manufacturer’s instructions for phosphoprotein arrays. (C) Ctx^R overexpress EGFR and have increased AKT signaling pathway. Ctx^S parental cells (HP) and Ctx^R clones (HC1, HC4, HC8) were harvested and protein lysates were fractionated on SDS-PAGE followed by immunoblotting for the indicated proteins. α-Tubulin was used as a loading control.