Reducing PHLPP1 phosphatase and pERK kinase activity rescues Runx2-mediated inhibition of pAkt in MDA-MB-231 cells. A) The PHLPP1 mRNA expression was transiently suppressed in MDA-MB-231 cells and gene expression was analyzed by real-time PCR. A quantification of GAPDH normalized PHLPP1 mRNA expression is shown. B) A quantification of the relative phosphorylation levels of pAkt (Serine 473) were determined by Western blotting in PHLPP1 knockdown MDA-MB-231 cells. C) The levels of pAkt (Serine 473) and total Akt proteins were analyzed by Western blot in PHLPP1 knock out control or Runx2 suppressed MDA-MB-231 cells as indicated. A quantification of pAkt levels normalized to total Akt protein is shown below the respective lane. D) The control or Runx2 knockdown MDA-MB-231 cells were stimulated with EGF for indicated times in the presence or absence of ERK inhibitor U0126. The level of pAkt and Akt was determined by Western blotting and quantification is shown below the respective lane. E) The Runx2 gene expression was conditionally suppressed by doxycycline in MDA-MB-231 cells stably expressing tTR-KRAB and Runx2-shRNA. The serum-deprived cells were stimulated with EGF in the presence of ERK inhibitor PD184161 for indicated times. The pAkt and total Akt expression was analyzed by Western blotting and a quantification of normalized expression is shown below the respective lanes.