Fig. 5: Low PTEN^S380D level causes AKT hyperactivity and PIN lesion formation.

a WB of lysates from 2-month-old prostates (n = 3 individual samples) of indicated genotypes. Blots are probed for PTEN and indicated phosphorylated PTEN. Ponceau S (PonS) staining served as loading control. b Tissue fractionation of prostates of the specified genotypes. Indicated fractions (C, cytoplasm; M, membrane; N, nuclear) were blotted and probed for PTEN (various exposures) and compartment-specific proteins. n = 3 independent fractionation experiments. c Measurements of PTEN and PTEN^S380D protein turnover rates in P4 MEFs by inhibiting protein synthesis with 20 µg/ml cycloheximide for the indicated times. PTEN levels at each timepoint were assessed by WB for PTEN, with Tubulin as loading control. N = 3 independent experiments. d Cell fractionation of MEFs of the indicated genotypes and fractions as in (b), blotted and probed for PTEN. Blots were overexposed to be able to visualize PTENα and PTENβ, n = 2 independent fractionation experiments. e As in (a), but now blots are probed for PTEN, AKT and AKT substrates, and are representative of at least 3 individual samples. f Percentage of prostate tubules with indicated PIN lesions at 6 months. A/–, Pten^S380A/–; D/–, Pten^S380D/–. N = 5 mice per genotype. Data are presented as mean values ± SEM. Statistical significance was assessed by ordinary one-way ANOVA, followed by Dunnett’s multiple comparisons test compared to +/+. g Immunostaining of consecutive sections of prostate tissue of indicated genotypes stained for PTEN and P-AKT^S473, n = 5 prostates per genotype. h Consecutive immunostaining of prostate tissue of indicated genotypes stained for PTEN and P-AKT^S473-Alexa Fluor 488 conjugate. N = 2 prostates per genotype, both stained at two different levels in two independent experiments. *Indicates lesions. Scale bars in (g and h) are 100 µm. Source data are provided as a Source Data file.