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LAF1 inhibits HFR1 ubiquitination in vitro. Epitope-tagged HFR1, LAF1, LAF1(R97A), and COP1 were expressed in E. coli, and in vitro ubiquitination assays were performed. COP1 (E3)-mediated ubiquitination of HFR1 was performed with increasing amounts of LAF1 (A) and LAF1(R97A) (B) and vice versa, and confirmed by Western blot analyses. Numbers indicate the relative amounts of proteins present in the reaction, where 1 represents 100 ng of MBP-HFR1-3HA, GST-LAF1, or GST-LAF1(R97A).
