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

Figure 2.

Figure 2.

Localization of the SIMs in ARIP4. (A) Interaction domain mapping of ARIP4. Schematic indicates the truncated forms of ARIP4 that were fused to GST. These GST-ARIP4s (right bottom panel) were incubated with in vitro sumoylated (right top panel) FLAG-Ad4BP/SF-1. Precipitates with glutathione Sepharose were immunoblotted with anti-FLAG antibody. Closed arrowheads indicate unsumoylated Ad4BP/SF-1, and single and double open arrowheads indicate Ad4BP/SF-1 that was sumoylated at one or two sites, respectively; 2% input is indicated. (B and C) The amino acids of ARIP4 responsible for its interaction with sumoylated Ad4BP/SF-1. Amino acid substitutions were made in the candidate sequences for the SIMs in the ARIP4 domains 2 and 5. Muts A to F and Muts 1–7 carry indicated amino acid substitutions (underlined). These proteins were interacted with sumoylated Ad4BP/SF-1 (bottom panels). Glutathione Sepharose precipitates were immunoblotted with anti-FLAG M2 antibody. The single and double open arrowheads indicate Ad4BP/SF-1 that was sumoylated at one or two lysine residues, respectively; 2% input is indicated. (D) The cooperative interaction through the two SIMs of ARIP4. As illustrated on the left, HA-tagged wild-type (WT) ARIP4 and ARIP4 carrying a V431A/I432A (mN-SIM) mutation, an E1463A/V1464A (mC-SIM) mutation, or both mutations (mNC-SIM) were prepared. These proteins were subjected to binding assays with sumoylated Ad4BP/SF-1. As indicated on the right, the anti-FLAG M2 agarose precipitates were immunoblotted with anti-HA antibody; 2% input is indicated.