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

Fig. 5.

Fig. 5.

COP1 degrades RUP1 and RUP2 under UV-B light. (A) Effect of COP1 on RUP2 stability in Arabidopsis under UV-B light. Immunoblot analysis of RUP2 proteins in 4-d-old Col and cop1-4 seedlings grown under +UV-B light and treated with 500 μM CHX and/or 50 μM MG132 for 3 h. RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. (B) Effect of COP1 on RUP2 stability in vitro under UV-B light, as analyzed by cell-free degradation assays. Purified His-RUP2 was incubated with total proteins extracted from 4-d-old Col and cop1-4 seedlings grown under +UV-B light for 2 h. The degradation mixture was treated with or without 50 μM MG132. His-RUP2 was detected with anti-RUP2 antibodies. RPN6 was used as a loading and negative control. (C) Effect of FLAG-COP1 on the ubiquitination of RUP2-HA in HEK293T cells. Total proteins were extracted from HEK293T cells that were transfected with FLAG/FLAG-COP1 and HA/RUP2-HA for co-IP with Dynabeads Protein G and anti-HA antibodies. Proteins were analyzed by immunoblotting with anti-HA and anti-Ubiquitin antibodies. Ubn, ubiquitin chain. The asterisks indicate nonspecific bands. (D and E) Effect of COP1 on the ubiquitination of FLAG-RUP1 in vivo. Total proteins were extracted from 4-d-old Col, FLAG-RUP1, FLAG-RUP1/cop1-4 (D), or FLAG-RUP1 YFP-COP1 (E) seedlings grown under +UV-B light and treated with 50 μM MG132 for 24 h before co-IP with ANTI-FLAG Magnetic Beads. Proteins were analyzed by immunoblotting with anti-FLAG and anti-Ubiquitin antibodies. The asterisks indicate nonspecific bands.