Cell-Free Protein Degradation Assay

PUB40-YFP bri1-5 and bri1-5 plants were ground into a powder with liquid N and proteins were extracted in degradation assay buffer (50 mM of Tris-Cl at pH 7.5, 50 mM of NaCl, 1% Triton X-100, 0.2 of mM phenylmethylsulfonyl fluoride, 5 mM of MgCl₂, 5 mM of DTT, and 5 mM of ATP). After centrifugation, an equal amount of each supernatant was incubated with 100 ng of MBP-BZR1 or MBP-bzr1-1D. To generate MBP-pBZR1, MBP-BZR1 that was preincubated with GST-BIN2 and ATP in kinase assay buffer (20 mM of Tris at pH 7.5, 1 mM of MgCl₂, 100 mM of NaCl, and 1 mM of DTT) was purified by affinity purification. After incubation for the indicated times at 30°C, the reactions were stopped by adding 2× SDS sample buffer.