Figure 1.

IDOL is conjugated with SUMO1 primarily at the K293 residue.A, alignment of the IDOL sequence across species with two inverted SUMOylation consensus motifs E/DxKψ (where x stands for any amino acid and ψ for a hydrophobic amino acid) shaded in gray. The K20 and K293 residues are in red. B–D, Huh7 cells were transfected with the indicated plasmids. After 48 h, cells were treated with 10 μM MG132 for 3 h and harvested. Lysates were pulled down by Ni-NTA agarose and then subjected to immunoblotting with the indicated antibodies. Asterisks indicate nonspecific bands. E, densitometric analysis of the high-molecular-weight smears relative to the unmodified IDOL band as shown in (D). Values are normalized to the value obtained from cells transfected with the wild-type form of IDOL only and presented as mean ± SEM (n = 3 independent experiments). ∗∗p < 0.01, ∗∗∗p < 0.001, ns, no significance. One-way ANOVA. Results in (B) and (C) are representative of two independent experiments and in (D) are of three independent experiments. IB, immunoblotting; IDOL, inducible degrader of the low-density lipoprotein receptor; SUMO, small ubiquitin-like modifier; WT, wild-type.