Confirmation of MDM2 substrates in mammalian cells by an <i>ex vivo</i> ubiquitination assay.

<p>HEK293T cells were transfected with the GFP-tagged substrates alone or co-transfected with Flag-tagged MDM2 (either WT or a catalytically inactive MDM2Δring mutant). The GFP-tagged substrate was immunoprecipitated from cell lysates with the anti-GFP antibody and immunoblotted with the anti-HA antibody to detect its ubiquitination. DDX42 (A) and TP53RK (B) were ubiquitinated when MDM2 was co-expressed. TP53RK and DDX42 were ubiquitinated when MDM2 was co-expressed, whereas only very faint ubiquitination bands were observed in the MDM2Δring lane. RPL36a (C) was ubiquitinated when MDM2 was co-expressed, while weaker ubiquitination bands were observed in the MDM2Δring lane. To calculate the relative extent of RPL36a poly-ubiquitination, the poly-ubiquitination signal of RPL36a (poly-Ub-RPL36a) was quantified using ImageJ software, divided by the corresponding GFP-RPL36a signal and normalized to 1.0. HEK293T (D) cells were transfected with empty vector of GV142 expressed only GFP protein (negative control) alone or with co-transfected Flag-tagged MDM2 (either WT or a catalytically inactive MDM2Δring mutant). The GFP was immunoprecipitated from cell lysates with the anti-GFP antibody and immunoblotted with the anti-HA antibody to detect its ubiquitination. GFP itself was not ubiquitinated by MDM2. HA-ubiquitin (E) and GFP-P53 (positive control) were co-transfected into HEK293T cells. The GFP-P53 was immunoprecipitated from cell lysates with the anti-GFP antibody and immunoblotted with the anti-HA antibody to detect its ubiquitination. P53 was ubiquitinated when MDM2 was co-expressed. GFP tagged TP53RK (F) or RPL36a (G) were transfected into HEK293T cells alone or co-transfected with Flag-tagged MDM2. The protein content of the lysates of the transfected cells were separated and immunoblotted using the anti-GFP antibody. The expression of MDM2 induced the significant degradation of transfected TP53RK and RPL36a. When the transfected cells were treated with 10 or 20 μM MG132, the degradation of TP53RK and RPL36a were blocked. *: ubiquitination bands.