Fig. 8. Mutations in the COP1 WD40 domain or in the interacting motif of HY5 interfere with dark-dependent degradation of HY5 in vivo. (A) Seedlings homozygous for both cop1-5 and transgenes expressing the indicated substituted COP1 protein were grown in continuous white light (200 µmol/m²/s) for 4 days and then transferred to darkness for 20 h. OE represents 35S-driven overexpression of wild-type COP1 in wild-type Columbia background. Protein extracts were prepared at day 4 (4 Day L) and after 20 h in darkness (20 h D), and the protein concentration was normalized. The amounts of HY5 and α-tubulin proteins were assayed by immunoblotting. (B) The 35S::HY5 and 35S::HY5VP-AA seedlings were grown in white light for 3 days and then transferred to darkness. Protein extracts were prepared at day 3 and after 24, 48 and 72 h in darkness, and the protein concentration was normalized. The amount of HY5 was assayed by immunoblotting, and the asterisk marks a cross-reacting band (Osterlund et al., 2000) that serves as internal loading control. Two independent lines of each transgene were examined and similar results were observed.