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. 2011 Aug 12;12(10):1018–1023. doi: 10.1038/embor.2011.152

Figure 2.

Figure 2

Krox20 mediates Nab sumoylation. (A–F) 293T cells were transfected with Flag–Nab or Flag–RanGAP1-C-ter (Flag–Ran) expression vectors and the constructs indicated at the top of each panel. Flag-tagged proteins were detected by western blot. Black arrowheads indicate non-modified proteins, and other arrowheads indicate modified proteins. Bottom panels correspond to inputs of the indicated proteins. (A,B) Nab is sumoylated in the presence of Krox20, and sumoylation is enhanced if low amounts of histidine-tagged small ubiquitin-like modifier 1 (His–SUMO1) or green fluorescent protein (GFP)–SUMO1 are transfected. (C) Nab is specifically sumoylated by Krox20, as the addition of NeuroM does not affect Nab sumoylation state; however, dominant-negative Ubc9 (C93S) interferes with Nab sumoylation. (D) The Nab K379RK517R (KR2) protein is not sumoylated. (E) Krox20 I268F and Nab Q64RH95Q mutants, affected in their ability to interact with each other, are ineffective as ligase and target in the sumoylation reaction, respectively. (F) Sumoylation of RanGAP1-C-ter is not altered by the presence of Krox20 or the I268F mutant. (G–I) Sumoylation of endogenous Nab was analysed in P19 cells in pull-down experiments with anti-Nab antibodies. (G) Endogenous Nab is sumoylated following His–SUMO1 transfection and serum stimulation, which results in the expression of Krox20. (H) Sumoylation of endogenous Nab was also observed after transfection of a Krox20 expression construct under normal growth conditions. (I) Nab sumoylation was prevented by transfection of Krox20 short interfering RNA (siRNA; si), but not by a control GFP siRNA, under serum stimulation conditions. Upper panels in G–I show pulled His–SUMO1–Nab, revealed with Nab antibodies, whereas lower panels show 1.5% input of the indicated proteins. Note that Krox20 antibodies also reveal a nonspecific upper band. (J) In vitro sumoylation assays with 300 ng of purified Flag-tagged Nab were carried out in the presence or the absence of mature SUMO (SUMO1GG) and 15 ng of Krox20 as indicated. Sumoylated products corresponded to 52.96%±2.74 (mean±s.d.) of loaded protein, as determined by chemiluminescence measurement from three independent western blots. HA, haemagglutinin epitope tag.