pcDNA3-Myc-His-Rap80 was used as the template to perform the in vitro translation (IVT) experiment with 35S-Met using the TnT Quick Coupled Transcription/Translation System (Promega) according to the manufacturer’s instructions. Two hundred microliters of 0.5 μM biotin-ds70mer DNA was incubated with 1 μg of K63-linked polyubiquitin (2 to 7) chains at room temperature for 2 hours and washed thoroughly to eliminate nonspecific binding. Increased amounts of IVT-Rap80 (10, 20, and 30 μl, respectively) were added and further incubated at 4°C for 2 hours before being washed thoroughly and resolved on SDS-PAGE for autoradiography.

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