Figure 3.

SENP1 deSUMOylates IDOL and reduces IDOL abundance.A, HEK293T cells were transfected with plasmids expressing Flag-tagged SENP1 or USP19. After 48 h, cells were harvested and IPed with anti-FLAG M2 agarose beads. Bound proteins were eluted by 3×FLAG peptides and analyzed by immunoblotting. B–C, equal amounts of SENP1 or USP19 in TBS (vehicle control) were incubated with 1 μM SUMO1-AMC (B) or 0.5 μM ubiquitin (Ub)-AFC (C), and released fluorescence of free AMC or AFC was analyzed. D, Huh7 cells were transfected as indicated. After 48 h, cells were treated with 10 μM MG132 for 3 h and harvested for the SUMOylation assay as described in Figure 1. Asterisk indicates nonspecific bands. E, Huh7 cells were transfected as indicated. After 48 h, cells were harvested for immunoblotting analysis. F, densitometric analysis of the IDOL bands in (E). Values are normalized to the value obtained from cells transfected with IDOL and UBC9 plus SUMO1 and presented as mean ± SEM (n = 3 independent experiments). ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns, no significance. One-way ANOVA. Results in (A), (B), and (C) are from a single experiment. Results in (D) are representative of two independent experiments and in (E) are of three independent experiments. AFC, 7-amino-4-trifluoromethylcoumarin; AMC, 7-amino-4-methylcoumarin; IB, immunoblotting; IDOL, inducible degrader of the low-density lipoprotein receptor; SENP, SUMO-specific peptidase; SUMO, small ubiquitin-like modifier; USP, ubiquitin-specific protease; WT, wild-type.