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. 2009 Oct 1;20(19):4235–4245. doi: 10.1091/mbc.E08-12-1247

Figure 1.

Figure 1.

Identification of proteins that preferentially interact with sumoylated Ad4BP/SF-1. (A) Affinity purification of proteins that interact with sumoylated Ad4BP/SF-1. FLAG-Ad4BP/SF-1 (lane 11) was conjugated with SUMO-1 in vitro (lane 12). The sumoylated and unsumoylated FLAG-Ad4BP/SF-1 were incubated with HeLa (lanes 1–3), HEK293 (lanes 4–6), or Y-1 (lanes 7–9) cell nuclear extracts. After recovery with anti-FLAG M2 agarose, the proteins were subjected to SDS-PAGE. The arrows in lanes 3, 6, and 9 indicate a protein that was specifically recovered by sumoylated Ad4BP/SF-1. (B) Affinity purification with SUMO-1– and -2–modified Ad4BP/SF-1. FLAG-Ad4BP/SF-1 (lane 6) was modified in vitro with either SUMO-1 (lane 7) or SUMO-2 (lane 8) and the products were incubated with HeLa nuclear extracts (NE) followed by pulldown with anti-FLAG M2 agarose (lanes 1–4). The recovered proteins were subjected to SDS-PAGE (top panel) and immunoblotting with anti-FLAG M2 antibody (bottom panel). Open and closed arrowheads indicate sumoylated and unsumoylated Ad4BP/SF-1, respectively. The arrows in lanes 3 and 4 indicate ARIP4. (C) Identification of ARIP4 by immunoblotting. The proteins recovered above were subjected to immunoblotting with anti-ARIP4 antibody. As indicated by the arrow, ARIP4 was recovered by both Ad4BP/SF-1 modified with SUMO-1 (lane 4) and SUMO-2 (lane 5), but not with unsumoylated Ad4BP/SF-1 (lane 3); 5% input is shown as a control (lane 1).