FIGURE 4.

AtSIZ1 is unstable and negatively regulated by COP1. (A) Degradation of AtSIZ1 by the proteasome pathway. Transgenic plants transformed with 35S-AtSIZ1-HA3 were treated with MG132, and AtSIZ1 levels were examined by Western blot analysis using an anti-HA antibody. Three different transgenic lines were used for the experiment. (B) Specificity of the anti-AtSIZ1 antibody. Total proteins were extracted from 2-weeks-old wild-type and siz1-2 mutant plants, and AtSIZ1 was detected by Western blot analysis using the anti-AtSIZ1 antibody. Tubulin was used as a loading control. (C) Effect of COP1 overexpression on AtSIZ1 levels. Two-weeks-old XVE-DN-COP1-Myc₆ transgenic plants were treated with β-estradiol, and AtSIZ1 levels were determined by Western blot analysis using an anti-AtSIZ1 antibody. Tubulin was used as a loading control. (D) Effect of COP1 on AtSIZ1 levels in vivo. Wild-type seedlings and cop1-4 mutants were grown for 4 days under light or dark conditions, and AtSIZ1 levels were determined by Western blot analysis using an anti-AtSIZ1 antibody. BIP (binding protein) was used as a loading control.