Figure 4.

NEDD4 activated Akt signaling pathway through diminished PTEN protein level in fibroblasts. (A) In vitro ubiquitination assay of PTEN by NEDD4. NEDD4 ubiquitinated PTEN directly. (B) NEDD4 ubiquitinated PTEN directly and promoted the protein degradation. Immunoblot analysis for endogenous PTEN in NEDD4 over-expressing NIH3T3 cells. Control or NEDD4 expression vector were transfected and incubated for 48 h. β-actin (ACTB) was blotted as the loading control. (C) Over-expression of NEDD4 in NIH3T3 cells enhanced the phosphorylation level of Akt. Control or NEDD4 expression vector were transfected and incubated for 48 h. Phosphor-Akt and total Akt were detected by immunoblotting. β-actin (ACTB) was blotted as the loading control.