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. 2019 Feb 20;116(10):4722–4731. doi: 10.1073/pnas.1816268116

Fig. 3.

Fig. 3.

CUL4-DDB1-RUP1/RUP2 E3 ligase mediates UV-B–induced degradation of HY5. (A) Phenotypes of 4-d-old Col, cul4cs, rup1-1 rup2-1, and cul4cs rup1-1 rup2-1 seedlings grown under −UV-B and +UV-B light conditions. (B) Hypocotyl length of the seedlings shown in A (mean ± SD, n ≥ 30). The asterisks indicate significant differences by Student’s t test (**P < 0.01) compared with Col under each light condition. (C) HY5 protein levels in 4-d-old Col, cul4cs, rup1-1 rup2-1, and cul4cs rup1-1 rup2-1 seedlings grown under −UV-B and +UV-B light conditions. Proteins were analyzed by immunoblotting with anti-HY5 and anti-RPN6 antibodies. RPN6 was used as a loading control. (D) Effect of RUP1 and RUP2 on HY5 stability in Arabidopsis under UV-B light. Immunoblot analysis of HY5 proteins in 4-d-old Col and rup1-1 rup2-1 seedlings grown under +UV-B light and treated with 500 μM CHX and/or 50 μM MG132 for 3 h. HY5 was detected with anti-HY5 antibodies. RPN6 was used as a loading and negative control. (E) Effect of CUL4, RUP1, and RUP2 on HY5 stability in vitro, as analyzed using cell-free degradation assays. Purified GST-HY5 was incubated with total proteins extracted from 4-d-old UV-B light-grown Col and cul4cs rup1-1 rup2-1 seedlings for 6 h. The degradation mixture was treated with or without 50 μM MG132. GST-HY5 was detected with anti-GST antibody. RPN6 was used as a loading and negative control. (F) Effect of FLAG-RUP1 on HY5 stability in vitro, as analyzed by cell-free degradation assays. Purified GST-HY5 was incubated with total proteins extracted from 4-d-old +UV-B light-grown Col and FLAG-RUP1 seedlings for 2 h. The degradation mixture was treated with or without 50 μM MG132. GST-HY5 was detected with anti-GST antibody. RPN6 was used as a loading and negative control. (G) Phenotypes of 4-d-old Col, rup2-1, FLAG-RUP2/rup2-1, and FLAG-mRUP2/rup2-1 seedlings grown under −UV-B and +UV-B light conditions. (H) Hypocotyl length of the seedlings shown in G (mean ± SD, n ≥ 30). The asterisks indicate significant differences by Student’s t test (**P < 0.01) compared with Col under each light condition. The mRNA levels (I) and protein levels (J) of HY5 in the seedlings shown in G are illustrated (mean ± SD, n = 3).