Fig. 1.

HY5 interacts with RUP1 and RUP2. (A) HY5 interacts with RUP1 in yeast two-hybrid assays. β-Galactosidase activity was quantified using o-nitrophenyl-β-d-galactopyranoside as a substrate (mean ± SD, n = 3). The asterisks indicate a significant difference by Student’s t test (**P < 0.01). (B) HY5 interacts with RUP1 and RUP2 in vitro. Purified GST or GST-HY5 was incubated with His-RUP1 or His-RUP2 before being pulled down by Glutathione Sepharose 4B. His-RUP1 and His-RUP2 were detected by anti-RUP1 and anti-RUP2 antibodies, respectively. (C) HY5 interacts with RUP1 and RUP2 in N. benthamiana, as assayed by firefly LCI. The color bar shows the range of luminescence intensity. The minus symbols (−) indicate empty vectors of nLUC or cLUC. (D) TAPa-HY5 associates with RUP2 in Arabidopsis. Total proteins were extracted from 4-d-old seedlings grown under −UV-B and +UV-B light conditions for co-IP with Anti–c-Myc Affinity Gel. Proteins were analyzed by immunoblotting with anti–c-Myc, anti-RUP2, and anti-RPN6 antibodies. RPN6 was used as a loading and negative control. (E) TAPa-HY5 associates with FLAG-RUP1 in Arabidopsis. Total proteins were extracted from 4-d-old seedlings grown under −UV-B and +UV-B light conditions for co-IP with Anti–c-Myc Affinity Gel. Proteins were analyzed by immunoblotting with anti–c-Myc, anti-FLAG, and anti-RPN6 antibodies. RPN6 was used as a loading and negative control.