Fig. 3.

The effects of FATS-262D/N on p53 ubiquitination and activation. a Purified GST protein or GST-tagged FATS protein was incubated with purified E1 and ubiquitin protein in ubiquitination buffer at 30 C for 90 min. The polyubiquitination was examined by Western blot using an ubiquitin-specific antibody (n = 3). FATS-262 N exhibited stronger E3 activity than did FATS-262D in assembling ubiquitin chains. b Purified GST protein or GST-tagged FATS protein was incubated with purified E1, ubiquitin and p53 protein in ubiquitination buffer at 30 °C for 90 min. The non-proteolytic polyubiquitination was examined by Western blot using a p53-specific antibody (n = 3). c MCF-7 cells were transfected with indicated vectors. Luciferase reporter assay was performed in triplicate in three independent experiments after transfection for 24 h. The pGL2-p21-luc vector contains a p21 promoter with p53-responsive elements. d MCF-7 cells were transfected with Flag-tagged FATS-262D or FATS-262 N or an empty vector, respectively. After 24 h, cells were treated with etoposide (25 μM) for the indicated time. Cell lysates were subjected to immunoblotting, and the results assessed quantitatively (n = 3). Ac, acetylation; Phos, phosphorylation