Figure 2
From: The E3 ubiquitin-protein ligase MDM2 is a novel interactor of the von Hippel–Lindau tumor suppressor
pVHL interacts with MDM2 is an isoform-specific manner. (A) Left panel: Schematic representation of pVHL30 and pVHL19 domains. The two isoforms differ for the presence of an intrinsically disordered N-terminus (N). Right panel: Y2H assay showing that pVHL30 specifically associates with MDM2. C + , positive control. C-, negative control (n = 10). (B) Immunoprecipitation (IP) of total protein extracts from HEK293T cells expressing Flag-MDM2 and HA-VHL30 with an anti-Flag antibody and immunostained with anti-Flag antibody, and anti-pVHL/anti-HA antibodies, as indicated. Arrow correspond to HA-VHL30 recognized by anti-VHL antibody (n = 3). (C) IP of total protein extracts from MN-1 cells expressing pVHL30-GFP, pVHL19-GFP, and soluble GFP, as indicated, and processed for pull down with anti-GFP antibody revealed endogenous MDM2 pull down only in the presence of pVHL30-GFP. Endogenous MDM2 was detected with a specific antibody. Asterisks indicate from left to right: pVHL30-GFP, pVHL19-GFP, and GFP both in input and Ip panels (n = 2). (D) IP of total protein extracts from HEK293T cells with either an anti-MDM2 antibody and IgG mouse as control or anti-pVHL antibody and IgG rabbit as control showed specific reciprocal pull down of endogenous proteins. Asterisks indicate the specific band corresponding to MDM2 protein (n = 3).
