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.