Figure 6.

E2F1 destabilizes RARα by promoting ubiquitination-mediated degradation of RARα. (A) MG132 blocks RARα degradation induced by ectopic expression of E2F1. U2OS cells were treated with or without 20 μM MG132 for 12 h; then the protein levels of RARα and E2F1 were determined. (B) E2F1 does not influence mRNA levels of RARα. The mRNA levels of RARα and E2F1 in U2OS cells transfected with E2F1 plasmid (left) or E2F1 siRNA (right) were determined. Expression levels were normalized to GAPDH. (C) Overexpression of E2F1 decreases half-time of RARα. U2OS cells were treated with 10 μg/ml cycloheximide (CHX), and then the protein levels of RARα and E2F1 were determined. The relative levels of RARα were normalized to β-actin as indicated (low). (D) Knocking down of E2F1 inhibits ubiquitination of RARα. U2OS cells were transfected with scramble or E2F1 siRNA followed by treatment with ATRA and MG132 for 12 h; then cell lysates were immunoprecipitated with anti-RARα and immunoblotted with anti-ubiquitin. (E) E2F1 inversely regulates RARα promoter activity. U2OS cells were transfected with RARE and Renilla reporter genes plus vector and E2F1 plasmids (left) or scramble and E2F1 siRNA (right) followed by treatment with 1 μM ATRA for 6 h. Firefly luciferase activities were measured and normalized to Renilla. (A and C) U2OS cells were transfected with vector and E2F1 plasmids. *, P < 0.05; **, P < 0.01.