Fig. 3. PTEN is required for Sema3A-mediated Akt/GSK-3 signalling in N1E-115 cells. N1E-115 cells were transfected with GFP, GFP-PTEN or GFP-PTEN C124S (GFP-PTEN CS) and stimulated with Sema3A (1 µg/ml) for 5, 10, 20 or 30 minutes (A,B,C). Western blots using P(Ser473)-Akt and P(Ser9)GSK-3ß antibodies reveal a decrease in phosphorylation following Sema3A treatment in GFP- and GFP-PTEN-expressing cells, but not in cells expressing GFP-PTEN C124S. (D) N1E-115 cells were transfected with a nonspecific siRNA (^*) or PTEN siRNA, and stimulated with Sema3A as previously. Knockdown of PTEN rendered NIE-115 cells insensitive to changes in GSK-3 and Akt phosphorylation in response to Sema3A. (E) In addition, the Sema3A response at 10 minutes (S) was compared with LY294002 treatments (LY, at 10 µM for 10 minutes). (F) Normalised relative band density (P-Akt/Akt, P-GSK-3/GSK-3) of each transfection experiment (mean + s.e.m. of three experiments).