Figure 3.

PRG2 Regulates pAKT and PI(3,4,5)P₃ in Dependence of PTEN

(A) PRG2 rescues decreased pAKT-levels induced by PTEN overexpression. HEK293 cells were triple transfected with AKT-GFP, GFP-PTEN (or control vector), and Prg2-Flag/Prg2ΔC-Flag (or control vector). Cell lysates were analyzed with indicated antibodies.

(B) Bars represent normalized pAKT(S473) over total AKT ratios across treatments ± SEM of n = 5 independent experiments. ^∗∗p < 0.01, ^∗∗∗p < 0.001 (one-way ANOVA with Bonferroni post hoc test).

(C) PRG2 rescues the loss of PI(3,4,5)P₃-accumulations induced by PTEN overexpression. U87MG cells transfected with PH-AKT-mRFP (red) and GFP, GFP-PTEN^C124S, GFP-PTEN (green, insets), and Prg2-Flag (white, insets). Panels show single confocal sections. Arrowheads indicate PH-AKT foci at plasma membranes (PM). Scale bar: 10 μm.

(D) Cells were scored for the presence of peripheral PH-AKT foci. Bars show the means ± SD of two independent experiments performed in duplicate, n ≥ 50. ^∗∗p < 0.01 (t test).

(E) PRG2 protects from the loss of peripheral F-actin accumulations induced by PTEN overexpression. Cells were stained with Phalloidin (F-actin, red top panels), anti-GFP (for detection of GFP or GFP-PTEN, green bottom panels) and anti-FLAG antibodies (for detection of PRG2-FLAG or PRG2ΔC-FLAG, white insets in lower magnification). Panels show maximum intensity confocal projections. Arrowheads indicate peripheral F-actin foci. Scale bar: 10 μm.

(F) Cells were scored for presence of peripheral F-actin foci. Bars show means ± SD of three independent experiments performed in duplicates, n ≥ 60. ^∗∗p < 0.01 (t test).