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. 2016 Apr 11;291(22):11619–11634. doi: 10.1074/jbc.M115.713370

FIGURE 2.

FIGURE 2.

hDREF increases the amount of SUMO-conjugated protein in vivo. A, 35S-labeled full-length hDREF was synthesized by a cell-free coupled in vitro transcription/translation reaction in the presence of [35S]methionine and subjected to GST pull-down using GST fusion proteins as indicated. As a positive control, GST-hDREF was used because hDREF forms a homodimer. The data are representative of two independent experiments with similar results. B, HeLa cells were cotransfected with HA-hDREF plasmid and Myc-SUMO-1, Myc-SUMO-2, Myc-SUMO-3, Myc-Ubc9, Myc-PIAS1, or Myc-Pc2 plasmid as indicated. At 24 h after DNA transfection, the cells were fixed with 3.7% PFA and stained using anti-Myc and anti-HA antibodies. Single confocal optical sections (n > 10) are shown. Scale bar, 5 μm. C, HeLa cells were transduced with lentivirus expressing shRNA against hDREF or scramble control. At 72 h after transduction, the cells were transfected with CFP-SUMO-1, CFP-SUMO-2, or CFP-SUMO-3 and cultured for an additional 24 h. Whole cell lysates were prepared, and protein samples (20 μg of protein) were analyzed by immunoblotting (IB) using anti-hDREF antibody and anti-GFP antibody. Two independent experiments validated >85% knockdown of hDREF by shRNA. The data are representative of two independent experiments with similar results. D, schematic representation of the hDREF structural domain and amino acid residues required for SUMO conjugation. Mutations resided in the N-terminal BED finger domain (C47A, C50A, C47A/C50A, H61A, and H71A), putative SUMOylation domain (ΔM360 and ΔL401), or the C-terminal hATC (hAT family C-terminal dimerization: pfam05699) domain (ΔATC). HeLa cells were transfected with plasmid expressing WT or mutant HA-hDREF as indicated. Whole cell lysates were prepared at 24 h after DNA transfection, and hDREF and endogenous proteins conjugated with SUMO-1 were analyzed by immunoblotting using anti-HA and anti-GFP antibodies, respectively. The data are representative of three independent experiments with similar results.