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

FIGURE 3.

FIGURE 3.

hDREF directs Mi2α SUMOylation in vivo. A, HeLa cells were cotransfected with HA-Mi2α with or without of FLAG-hDREF and CFP-SUMO-1 plasmids as indicated. Whole cell lysates were prepared at 24 h after DNA transfection, and HA-Mi2α, FLAG-hDREF, and CFP or CFP-SUMO-1 were detected by immunoblotting analysis (IB) using anti-HA, anti-FLAG, and anti-GFP antibodies, respectively. The data are representative of two independent experiments with similar results. B, HeLa cells were cotransfected with HA-Mi2α and CFP-SUMO paralogs with or without FLAG-hDREF plasmid as indicated. Whole cell lysates were prepared at 24 h after DNA transfection and HA-Mi2α and CFP or CFP-SUMOs were detected by immunoblotting analysis using anti-HA and anti-GFP antibodies, respectively. The data are representative of two independent experiments with similar results. C, Mi2α is SUMOylated at Lys-1971 by hDREF. Left, alignment of putative SUMOylation sites on Mi2α. Right, HeLa cells were cotransfected with HA-Mi2α WT or KR mutants and CFP-SUMO-1 with or without FLAG-hDREF plasmid. At 24 h after transfection, whole cell lysates were prepared, and HA-Mi2α was detected by immunoblotting analysis using anti-HA antibody. The data are representative of two independent experiments with similar results. D, HeLa cells were cotransfected with HA-Mi2α and CFP-SUMO-1 with or without FLAG-hDREF (shRNA-resistant) expression plasmid and plasmid expressing shRNA against hDREF mRNA (KD) or scramble shRNA plasmid as indicated. Whole cell lysates were prepared at 24 h after DNA transfection, and HA-Mi2α was detected by immunoblotting analysis using anti-HA antibody. Knockdown of endogenous hDREF and expression of shRNA-resistant hDREF were evidently detected on an immunoblot by using anti-hDREF antibody. The data are representative of two independent experiments with similar results. E, HeLa cells were expressed with HA-Mi2α, CFP-SUMO-1, and WT or SUMOylation-defective hDREF mutants (W500A/W501A and LLVL/AAAA) as indicated. Whole cell lysates were prepared at 24 h after DNA transfection, and Mi2α polypeptides were detected by immunoblotting analysis using anti-HA antibody. The data are representative of two independent experiments with similar results.