Fig. 4.

Lipid phosphatase activity of PTEN regulates MOB1 protein level in vitro. a Neuro2A cells were transfected with Flag-PTEN or vector constructs. After 48 h of transfection, cells were lysed and 5% of lysate was used as input control and the remaining lysate was subjected to IP with an anti-Flag antibody. Input and IP were analyzed by western blotting. b, c Immunoblotting (IB) of PTEN, p-GSK3β (S9), GSK3β and MOB1 in Neuro2A cells transduced with WT or mutant PTEN (C124S, Y138L, or G129E). GAPDH was used as a loading control (*P < 0.05 vs. sh-PTEN group, ^#P < 0.05 vs. sh-EGFP group, ANOVA test followed by Dunnett’s post hoc test). d–l Stable PTEN-knockdown PC12 and Neuro2A cells were treated with specific pharmacological inhibitors of PTEN-PI3K downstream pathways for 2 h: 20 μM LY294002 (PI3K inhibitor) (d–f), 2 μM BX795 (PDK1 inhibitor) (g–i) and 20 μM Perifosine (Akt inhibitor) (j–l). Levels of p-PDK1 (S241), PDK1, p-Akt (T308), Akt, p-GSK3β (S9), GSK3β, MOB1,NF200 and PTEN were determined by IB. GAPDH served as an internal control (*P < 0.05 vs. sh-PTEN group, ^#P < 0.05 vs. sh-EGFP group, ANOVA test followed by Dunnett’s post hoc test)