FIGURE 2.

Flowering Locus C (FLC) is sumoylated by HPY2 in vitro. Arabidopsis His₆-AtSAE1b, His₆-AtSAE2, His₆-AtSCE1, GST-HPY2, His₆-AtSUMO1, and GST-FLC-Myc were overexpressed in E. coli and purified with Ni²⁺-NTA or glutathione affinity columns as appropriate. (A) Sumoylation of GST-FLC-Myc was assayed in the presence or absence of E1 (His₆-AtSAE1b and His₆-AtSAE2), E2 (His₆-AtSCE1), E3 (GST-HPY2), and His₆-AtSUMO1. FLC sumoylation was detected by Western blotting with anti-Myc antibody. (B) To identify the sumoylation site on FLC, GST-FLCm1-Myc (K54R), GST-FLCm2-Myc (K135R), and GST-FLCm3-Myc (K154R) were overexpressed in E. coli and purified using a glutathione affinity column. The reaction mixture contained E1 (His₆-AtSAE1b and His₆-AtSAE2), E2 (His₆-AtSCE1), E3 (GST-HPY2), and His₆-AtSUMO1 without (-) or with (+) a mutant protein instead of GST-FLC-Myc. FLC sumoylation was detected by Western blotting with anti-Myc antibody.