4.6 Protein extraction and western blot analysis
For protein extraction, about 50 mg of tissue samples were placed in a 2 ml plastic centrifugal pipe and grinded into fine powder in liquid nitrogen, then added 150 μL protein loading buffer (5% sodium dodecyl sulphate, 0.25% bromophenol blue, 2.5% β-mercaptoethanol, 25% glycerol) to the powder and boiled at 100°C for 10 min followed by centrifugation at 13000 g for 10 min at 4°C. Clear supernatants was used as total protein extract. The extracted protein solution was electrophoresed on 10% sodium dodecyl sulphate (SDS)-gel and transferred to the nitrocellulose membrane to incubate with antibody. For BES1 protein accumulation analysis, Proteins from different samples were detected by anti-BES1 (Yin’s lab) at a dilution of 1:5000.