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. 2014 May 16;26(5):1981–1991. doi: 10.1105/tpc.114.124024

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Figure 8. — CSU1 Ubiquitinates COP1.

(A) Ubiquitination assays were performed using UBE1 (E1), UbcH5b (E2), and HA-tagged ubiquitin (HA-Ub). Recombinant 6× His-CSU1 and MBP-COP1 were preincubated with 20 μM zinc acetate. MBP, nonincubated 6× His-CSU1 (-Zn), and MBP-COP1 (-Zn) were used as negative controls. Ubiquitinated MBP-COP1 was detected using anti-HA antibody. Asterisk indicates ubiquitinated MBP-COP1 (anti-HA). Values were calculated using ImageJ software.

(B) Immunoblots against ubiquitin showing that loss of CSU1 results in the reduction of COP1 ubiquitination in vivo. COP1 protein was immunoprecipitated by COP1 antibody and then blotted with monoclonal ubiquitin antibody (anti-Ub). Wild-type and csu1 seedlings were grown in the dark for 5 d. cop1-4 was used as a negative control. IP, immunoprecipitation.