FIGURE 5.

Identification and functional analysis of SIM. A, MTF-1-His₆ or constructs with the SIM mutation were co-transfected with SUMO-1 into CHO K1 cells. The His-tagged proteins were isolated with metal affinity beads and the levels of sumoylation on SIM mutants were analyzed with Western blotting. B, MTF-1-His₆ or constructs with the SIM mutation were co-transfected with FLAG-SUMO-1(AA) plasmid. The complexes were isolated by immunoprecipitation (IP) with anti-SUMO-1 antibodies and then subjected to Western blotting (IB) analysis. C, sumoylation-defective MTF-1, K627R, and E629A, and their SIM mutants were co-transfected with FLAG-SUMO-1(AA). Twenty-four h after transfection, the cells were harvested and cell lysates were prepared and subjected to immunoprecipitation with anti-SUMO-1 antibodies. The precipitated products were analyzed with anti-His₆ and anti-FLAG antibodies. D, MTF-1 plasmid or constructs with the SIM mutation were co-transfected with 5×MREd-luciferase reporter plasmids into CHO K1 cells. Luciferase activity was determined after exposing the cells with 100 μm zinc for 6 h. E, HEK293 cells were transfected with MTF-1 or SIM-3A plasmids, and treated with or without 100 μm zinc for 6 h before being harvested. Total RNA was extracted and the relative MTIIA mRNA level was determined by real-time PCR. Each value represents a mean ± S.D. of three samples. Asterisks denote significant differences (p < 0.05) with the MTF-1 (D) or between the paired samples (E).