Figure 5

Western blot of microsomal BnDGAT1 before (lane 1) and after Proteinase K treatment (lane 2). Microsomes were prepared from microspore-derived cell suspension cultures of B. napus L. cv Jet Neuf according to Byers et al. [27]. Microsomes containing 1 mg protein were treated for 30 min on ice with 10 μg Proteinase K. The hydrolytic reaction was terminated with 0.1 mg phenylmethylsulfonyl fluoride per mg Proteinase K and incubated on ice for a further 5 min according to Moromoto et al. [51]. Samples were diluted to 1 mL with 10 mM HEPES-NaOH (pH 7.4) and centrifuged for 20 min at 220,000 g at 4°C. The pellet was treated with 150 μl SDS-PAGE sample buffer and boiled for 2 min, and 25 μL were subjected to SDS-PAGE (12% total monomer and 1.1% cross-linker concentration).