Figure 3.

SPA1 promotes down-regulation of HY5 protein levels without affecting COP1 expression. Anti-HY5 immunoblot analyses of seedlings grown under continuous far-red (A,C) and 3-day-old seedlings transferred from darkness to far-red light (B) for indicated times. Immunoblots probed with anti-Rpt5 or anti-CSN3 antibodies are shown as loading controls. A Northern blot was probed with the ³²P-labeled HY5 cDNA. (D) COP1 levels in the 4-day-old continuous far-red-grown seedlings are indicated by the arrowhead. The asterisks indicate the positions of cross-reacting bands by the COP1 antibodies. The wild-type (WT) ecotype used (A,B,D) is RLD. Yeast two-hybrid analyses of the SPA1-HY5 interactions (E) and the SPA1-COP1 interactions (F,G). The constructs used for analysis are shown on the left. (Zn) RING-finger domain; (CC) coiled-coil domain; (WD40) WD40 repeat region; (SPA1-NT696) amino acids 1-696; (SPA1NT545) amino acids 1-545; (SPA1-CC) amino acids 521-696; (SPA1-CT509) amino acids 521-1029; (SPA1-CC) amino acids 546-646. The right side of the panel shows corresponding β-galactosidase activities. Error bars represent the standard deviation.