Figure 5. PTEN overexpression prevents serum-induced Akt phosphorylation after 15 min (A) and 30 min (B) serum induction using 10% FCS as determined by western blotting using an anti-pAkt-antibody.

An antibody against total Akt was used as control. The potent PI3-K-inhibitor Ly294002 (50 nM) was supplemented to the medium for not-transfected cells. HcASMC were transfected as follows: non transfected (NT); transfected with a plasmid coding for GFP (pControl); transfected with an empty plasmid without PTEN-cDNA (pControl); transfected with a plasmid coding for PTEN (pPTEN); mock transfected (without plasmid, but with transfection reagent). PTEN- and Akt co-overexpression reverses PTENs pro-apoptotic effect (C). HcASMC were co-transfected with a WT form of PTEN and a constitutively active form of Akt. An empty plasmid was used as control. Following the magnetic separation of positively transfected SMC, apoptosis was determined after cell growth for 24 h and expressed as mean OD405 ± SD using a cell death detection ELISA (^# P<0.001, *P<0.001; n = 4).