Figure 3.

Effects of Ubc domains of TSG101 on MDM2 degradation and ubiquitination. (a) The indicated constructs expressing c-Myc-tagged full-length and deletion mutants of TSG101 (A-F, 8 μg), a construct expressing HA-tagged MDM2 (1.5 μg), and a construct expressing GFP (2 ng) were introduced into U2OS cells by transfection. HA-MDM2, c-Myc-tagged TSG101s (A-F), and GFP were detected by Western blotting 48 h after transfection with anti-HA, anti-c-Myc, and anti-GFP antibodies. (b) The indicated constructs express ubiquitin tagged with both His₆ and c-Myc (HM-Ub, 5 μg, Left), or a dominant-negative variant ubiquitin tagged with His₆ and c-Myc (HM-K48R-Ub, 5 μg, Right). These were cotransfected into SJSA-1 cells with a GFP expression construct (2 ng), constructs expressing TSG101 mutant B or F (4 μg each), a construct expressing antisense TSG101 (4 μg), or a construct containing no DNA insert (4 μg). Protein extracts were applied to Ni-NTA columns, and the ubiquitin-labeled MDM2 was eluted and detected by Western blotting with anti-MDM2 antibody. An aliquot (1/20) of protein extracts was used for the detection of GFP by Western blotting to normalize for transfection efficiency.